Stable non-hygroscopic crystalline form of N-[N-[N-4-(piperidin-4-yl)butanoyl)-N-ethylglycyl] aspartyl]-L-β-cyclohexyl alanine amide

ABSTRACT

The invention is directed to a non-hygroscopic stable crystalline form of the antithrombotic compound N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanine amide, to processes for preparing said stable crystalline form, to a pharmaceutical composition thereof, and intermediates thereof, and the invention is directed also to processes for preparing a compound of the formula ##STR1## wherein: A, B, Z, E 1 , E 2 , G, R, m, n, and p are as defined herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of International Patent Application No.PCT/US97/14756, filed Aug. 21, 1997, which claims benefit of U.S. Ser.No. 60/024,284 filed Aug. 21, 1996, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to a non-hygroscopic stable crystalline formofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide of formula I. The compound has antithrombotic activity, ##STR2##including the inhibition of platelet aggregation and thrombus formationin mammals, and is useful in the prevention and treatment of thrombosisassociated with disease states such as myocardial infarction, stroke,peripheral arterial disease and disseminated intravascular coagulation.

In addition, the invention is directed to processes for preparing thecrystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide, a pharmaceutical composition thereof and intermediates thereof.

Haemostasis, the biochemistry of blood coagulation, is an extremelycomplex phenomena whereby normal whole blood and body tissuespontaneously and arrest bleeding from injured blood vessels. Effectivehaemostasis requires the combined activity of vascular, platelet andplasma factors as well as a controlling mechanism to prevent excessiveclotting. Defects, deficiencies, or excesses of any of these componentscan lead to hemorrhagic or thrombotic consequences.

Platelet adhesion, spreading and aggregation on extracellular matricesare central events in thrombus formation. These events are mediated by afamily of adhesive glycoproteins, i.e., fibrinogen, fibronectin, and vonWillebrand factor. Fibrinogen is a co-factor for platelet aggregation,while fibronectin supports platelet attachments and spreading reactions,and von Willebrand factor is important in platelet attachment to andspreading on subendothelial matrices. The binding sites for fibrinogen,fibronectin and von Willebrand factor have been located on the plateletmembrane protein complex known as glycoprotein IIb/IIa.

Adhesive glycoproteins, like fibrinogen, do not bind with normal restingplatelets. However, when a platelet is activated with an agonist such asthrombin or adenosine diphosphate, the platelet changes its shape,perhaps making the GPIIb/IIIa binding site accessible to fibrinogen. Thecompound within the scope of the present invention blocks the fibrinogenreceptor, and thus has the aforesaid antithrombotic activity.

2. Reported Developments

It has been observed that the presence of Arg-Gly-Asp (RGD) is necessaryin fibrinogen, fibronectin and von Willebrand factor for theirinteraction with the cell surface receptor (Ruoslahti E., Pierschbacher,Cell 1986, 44, 517-18). Two other amino acid sequences also seen to takepart in the platelet attachment function of fibrinogen, namely, theGly-Pro-Arg sequence, and the dodecapeptide,His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val sequence. Smallsynthetic peptides containing the RGD or dodecapeptide have been shownto bind to the platelet GPIIb/IIIa receptor and competitively inhibitbinding of fibrinogen, fibronectin and von Willebrand factor as well asinhibit aggregation of activated platelets (Plow, et al., Proc. Natl.Acad. Sci. USA 1985, 82, 8057-61; Ruggeri, et al., Proc. Natl. Acad.Sci. USA 1986, 5708-12; Ginsberg, et al., J. Biol. Chem. 1985, 260,3931-36; and Gartner, et al., J. Biol. Chem. 1987, 260, 11,891-94).

Indolyl compounds containing guanidinoalkanoyl- andguandinoalkenoyl-aspartyl moieties are reported to beplatelet-aggregation inhibitors by Tjoeng, et al., U.S. Pat. Nos.5,037,808 and 4,879,313.

U.S. Pat. No. 4,992,463 (Tjoeng, et al.), issued Feb. 12, 1991,discloses generically that a series of aryl and aralkyl guanidinoalkylpeptide mimetic compounds exhibit platelet aggregation inhibitingactivity and discloses specifically a series of mono- and dimethoxyphenyl peptide mimetic compounds and a biphenylalkyl peptide mimeticcompound.

U.S. Pat. No. 4,857,508 (Adams, et al.), issued Aug. 15, 1989, disclosesgenerally that a series of guandinoalkyl peptide derivatives containingterminal aralkyl substituents exhibit platelet aggregation inhibitingactivity and discloses specifically a series of O-methyl tyrosine,biphenyl, and naphthyl derivatives containing a terminal amidefunctionality.

Haverstick, D. M. et al., in Blood 66 (4), 946-952 (1985), disclose thata number of synthetic peptides, including arg-gly-asp-ser andgly-arg-gly-asp-ser, are capable of inhibiting thrombin-induced plateletaggregation.

Plow, E. F. et al., in Proc. Natl. Acad. Sci. USA 79, 3711-3715 (1982),disclose that the tetrapeptide glycyl-L-prolyl-L-arginyl-L-prolineinhibits fibrinogen binding to human platelets.

French Application No. 68/17507, filed Dec. 15, 1986, discloses thattetra-, penta- and hexapeptide derivatives containing the -arg-gly-asp-sequence are useful as antithrombotics.

U.S. Pat. No. 4,683,291 (Zimmerman, et al.), issued Jul. 28, 1987,discloses that a series of peptides, comprised of from six to fortyamino acids, which contain the sequence -arg-gly-asp- are plateletbinding inhibitors.

European Application Publication No. 0 319 506, published Jun. 7, 1989,discloses that a series of tetra-, penta-, and hexapeptide derivativescontaining the -arg-gly-asp- sequence are platelet aggregationinhibitors.

Cyclic peptide analogues containing the moiety Gly-Asp are reported tobe fibrinogen receptor antagonists in U.S. Pat. No. 5,023,233.

Peptides and pseudopeptides containing amino-, guanidino-, imidizaloyl,and/or amidinoalkanoyl, and alkenoyl moieties are reported to beantithrombotic agents in pending U.S. application Ser. Nos. 07/677,066,07/534,385, and 07/460,777 filed on Mar. 28, 1991, Jun. 7, 1990, andJan. 4, 1990, respectively, as well as in U.S. Pat. No. 4,952,562, andin International Application No. PCT/US90/05448, filed Sep. 25, 1990,all assigned to the same assignee as the present invention.

Peptides and pseudopeptides containing amino- and guanidino- alkyl- andalkenyl-benzoyl, phenylalkanoyl, and phenylalkenoyl moieties arereported to be antithrombotic agents in pending U.S. application Ser.No. 07/475,043, filed Feb. 5, 1990, and in International Application No.PCT/US91/02471, filed Apr. 11, 1991, published as InternationalPublication No. WO 92/13117 Oct. 29, 1992, assigned to the same assigneeas the present invention.

Alkanoyl and substituted alkanoyl azacycloalkylformyl aspartic acidderivatives are reported to be platelet aggregation inhibitors in U.S.Pat. No. 5,053,392, filed Dec. 1, 1989, and assigned to the sameassignee and having the same inventorship as the present invention.

N-substituted azacycloalkylcarbonyl cyclic aminoacylaspartic acidderivatives are reported to be antithrombotics in U.S. Pat. No.5,064,814, filed Apr. 5, 1990 by the same inventors and assigned to thesame assignee as the present invention. Azacycloalkylformylglycolaspartic acid derivatives are reported to be antithrombotics in U.S.Pat. No. 5,051,405, filed Oct. 10, 1989, and assigned to the sameassignee as the present invention.

European Patent Application 0 479 481, published Apr. 8, 1992, disclosesazacycloalkylalkanoyl glycyl aspartyl amino acids as fibrinogen receptorantagonists.

European Patent Application 0 478 362, published Apr. 1, 1992, disclosesazacycloalkylalkanoyl peptidyl β-alanines as fibrinogen receptorantagonists.

PCT Patent Application Publication No. WO95/10295 disclosesazacycloalkylalkanoyl peptides and pseudopeptides of formula II and, in##STR3## particular,N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide that inhibit platelet aggregation and thrombus formation inmammals and are useful in the prevention and treatment of thrombosis.TheN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared according to PCT Patent Application Publication No.WO95/10295 is amorphous, hygroscopic and is physically unstable as itabsorbs moisture. PCT Patent Application Publication No. WO95/10295 doesnot disclose a non-hygroscopic stable crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide.

PCT Patent Application Publication No. WO95/10295 also discloses thatthe azacycloalkylalkanoyl peptides and pseudopeptides are preparedgenerally by standard solid phase or solution phase peptide synthesisprocedures using starting materials and/or readily availableintermediates from chemical supply companies such as Aldrich or Sigma,(H. Paulsen, G. Merz, V. Weichart, "Solid-Phase Synthesis ofO-Glycopeptide Sequences", Angew. Chem. Int. Ed. Engl. 27 (1988); H.Mergler, R. Tanner, J. Gosteli, and P. Grogg, "Peptide Synthesis by aCombination of Solid-Phase and Solution Methods I: A New VeryAcid-Labile Anchor Group for the Solid-Phase Synthesis of FullyProtected Fragments. Tetrahedron letters 29, 4005 (1988); Merrifield, R.B., "Solid Phase Peptide Synthesis after 25 Years: The Design andSynthesis of Antagonists of Glucagon", Makromol. Chem. Macromol. Symp.19, 31 (1988)). Furthermore, PCT Patent Application Publication No.WO95/10295 discloses that the amorphous and hygroscopic form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide is prepared by sequential synthesis from the C-terminus amino acidas shown in Scheme I. PCT Patent ##STR4## Application Publication No.WO95/10295 does not disclose the formation oftetra-azacycloalkylalkanoyl peptides and pseudopeptides or, inparticular,N-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide from a central di(pseudopeptide or peptide) whereby the N- andC-Terminal ends of the central di(pseudopeptide or peptide) are bothcoupled with pseudoamino acids and/or aminoacids to form thetetra-azacycloalkylalkanoyl peptides and pseudopeptides.

SUMMARY OF THE INVENTION

The present invention is directed to a non-hygroscopic stablecrystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide of formula I. The compound has antithrombotic ##STR5## activity,including the inhibition of platelet aggregation and thrombus formationin mammals, and is useful in the prevention and treatment of thrombosisassociated with disease states such as myocardial infarction, stroke,peripheral arterial disease and disseminated intravascular coagulation.The invention is also directed to a pharmaceutical composition of thenon-hygroscopic stable crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide and intermediates thereof.

The invention is also directed to processes for preparing atetra-azacycloalkylalkanoyl peptide or pseudopeptide compound of formulaII ##STR6## wherein:

A is H;

B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl,alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl, or alkylaralkyl;##STR7##

Z is

E¹ is H;

E² is the a-carbon side chain of a naturally occurring a-amino acid, H,alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl,alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituedaralkyl, heterocyclyl, substituted heterocyclyl, heterocyclyalkyl,substituted heterocyclyalkyl, or E¹ and E² taken together with thenitrogen and carbon atoms through which E¹ and E² are linked form a 4-,5-, 6-, or 7-membered azacycloalkane ring;

G is OR¹ or NR¹ R² ;

R¹ and R² are independently H, alkyl, cycloalkyl, cycloalkylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl, oralkylaralkyl;

R is H, alkyl, aryl, or aralkyl;

m is 1 to 5;

n is 0 to 6; and

p is 1 to 4,

and, in particular, the non-hygroscopic stable crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a x-ray powder diffraction graph of a sample of thenon-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared in Example 13, Method A.

FIG. 2 represents a x-ray powder diffraction graph of a sample of thenon-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared in Example 13, Method B(a).

FIG. 3 represents a x-ray powder diffraction graph of a sample of thenon-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared in Example 13, Method B(b).

FIG. 4 represents a x-ray powder diffraction graph of a sample of thenon-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared as noted in Example 14.

FIG. 5 represents a x-ray powder diffraction graph of a sample of thenon-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared as noted in Example 14.

FIG. 6 represents isothermal microcalorimetric graph of the power outputas a function of time for three different experiments which areundertaken as described in Experiment 15. The experiments monitor thethermal activity of different crystalline forms ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide when exposed to various solvent vapors. The (A) trace in FIG. 6shows that a strong exothermic event takes place when hygroscopicN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared according to Examples 5 or 11 is exposed to 80% RH(saturated KCl solution) at 40° C. over 30 hours, during which exposurethe hygroscopic form of the compound is converted to the non-hygroscopicform of the compound. The (B) trace in FIG. 6 shows no exothermicconversion event takes place when hygroscopicN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared according to Examples 5 or 11 is exposed to methanolvapors at 40° C. (a solvent other than water in which the compound issoluble), and thus that methanol does not support mobility within thecrystals of that form for the conversion to the non-hygroscopic form.The (C) trace in FIG. 6 shows no exothermic conversion event takes placewhen non-hygroscopicN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared according to Example 13 is exposed to 40° C./80% RH, andthus that the non-hygroscopic form of the compound does not undergo aform conversion at those conditions, i.e., it is a stable form.

FIG. 7 represents isothermal microcalorimetric graph of the power outputas a function of time for three different experiments which areundertaken as described in Experiment 15. The experiments monitor thethermal activity of the conversion of the hygroscopic crystalline formofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide to its nonhygroscopic form when exposed to 80% RH at 40° C., 50°C. and 60° C. The Figure represents that the conversion takesapproximately 24 hours at 40° C., 6.5 hours at 50° C. and 3 hours at 60°C.

FIG. 8 represents isothermal microcalorimetric graph of the power outputas a function of time for four different experiments which areundertaken as described in Experiment 15. The experiments monitor thethermal activity of the conversion of the hygroscopic crystalline formofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide to its non-hygroscopic form when exposed at 60° C. to 65% RH, 75%RH, 80% RH and 100% RH. A salient features of FIG. 8 is that higherrelative humidities produce a faster conversion. Another salient featureis that the conversion to the non-hygroscopic form of the compoundoccurs at 100% RH at 60°° C. without the liquification which occurs tothe hygroscopic form at room temperature. Based on these results it isexpected that the rate of conversion to the non-hygroscopic form is muchfaster than the rate of liquification of hygroscopic form at 60° C.

FIG. 9 represents a comparison of the % weight gain vs. % RH plots forthe hygroscopic (▪) and non-hygroscopic () forms ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide at 25° C. which are undertaken as described in Experiment 16. FIG.9 represents that the hygroscopic form picks up more water than thenon-hygroscopic form as the RH increases, and more pronounced atrelative humidities greater than 60%. Furthermore, FIG. 9 representsthat the hygroscopic form of the compound does not desorb to itsoriginal weight % whereas the non-hygroscopic form of the compound odesdesorb to its original weight %.

DETAILED DESCRIPTION OF THE INVENTION

As used above, and throughout the description of this invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

The following abbreviations used herein include:

BOC (t-butyloxycarbonyl), CBZ (benzyloxycarbonyl), Gly (glycine), Asp(aspartic acid), Obzl (Benzyloxy), TFA (trifluoroacetic acid), Cha(β-cyclohexyl-alaline), EtOAc (ethyl acetate), DMF (dimethyl formamide),DCC (dicyclohexylcarbodiimide), HOBT) (hydroxybenzotriazole), TBTU(2-1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),DI (deionized water), PNP (p-nitrophenol), PFP (pentafluorophenol), DCU(dicyclohexyl urea), NMM (N-methylmorpholine), MTBE (methyl t-butylether), RH (relative humidity), THF (tetarhydrofuran) PipBu(4-piperidinebutyric acid) and PipBuen((4-Piperidin)butylidenylcarboxylic acid) is a compound of the formula##STR8##

"Patient" includes both human and other mammals.

"Pharmaceutically acceptable salt" means a salt form of the parentcompound of formula I which is relatively innocuous to a patient whenused in the therapeutic doses so that the beneficial pharmaceuticalproperties of the parent compound of formula I are not vitiated byside-effects ascribable to a counter ion of that salt form.Pharmaceutically acceptable salt also includes a zwitterion or internalsalt of the compound of formula I.

"Alkyl" means a saturated aliphatic hydrocarbon group which may bestraight or branched and having about 1 to about 20 carbon atoms in thechain. Branched means that a lower alkyl group such as methyl, ethyl orpropyl is attached to a linear alkyl chain. Preferred straight orbranched alkyl groups are the "lower alkyl" groups which are those alkylgroups having from 1 to 10 carbon atoms. Most preferred lower alkylgroups have from 1 to about 6 carbon atoms.

"Cycloalkyl" means a saturated carboxylic group having one or more ringsand having about 3 to about 10 carbon atoms. Preferred cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and decahydronaphthyl.

"Cycloalkyalkyl means an alkyl group substituted with a cycloalkylgroup. Preferred cycloalkylalkyl groups include cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, decahydronaphth-1-ylmethyl anddecahydronaphth-2-ylmethyl.

"Alkylcycloalkyl" means an cycloalkyl group substituted with an alkylgroup. Exemplary alkylcycloalkyl groups include 1-, 2-, 3-, or 4-methylor ethyl cyclohexyl.

"Alkylcycloalkylalkyl" means an alkyl group substituted by analkylcycloalkyl group. Exemplary alkylcycloalkyl groups include 1-, 2-,3-, or 4-methyl or ethyl cyclohexylmethyl or 1-, 2-, 3-, or 4-methyl orethyl cyclohexylethyl.

"Azacycloalkane" means a saturated aliphatic ring containing a nitrogenatom. Preferred azacycloalkanes includes pyrolidine and piperidine.

"Naturally occurring α-amino acid" means glycine, alanine, valine,leucine, isoleucine, serine, threonine, phenylalanine, tyrosine,tryptophan, cysteine, methionine, proline, hydroxyproline, asparticacid, asparagine, glutamine, glutamic acid, histidine, arginine,orthinine, and lysine.

"α-carbon side chain of a naturally occurring α-amino acid" means themoiety which substitutes the α-carbon of a naturally occurring α-aminoacid. Exemplary α-carbon side chains of naturally occurring α-aminoacids include isopropyl, methyl, and carboxymethyl for valine, alanine,and aspartic acid, respectively.

The term "amine protecting group" means an easily removable group whichis known in the art to protect an amino group against undesirablereaction during synthetic procedures and to be selectively removable.The use of amine protecting groups is well known in the art forprotecting groups against undesirable reactions during a syntheticprocedure and many such protecting groups are known, cf, for example, T.H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2ndedition, John Wiley & Sons, New York (1991), incorporated herein byreference. Preferred amine protecting groups are acyl, including formyl,acetyl, chloroacetyl, trichloracetyl, o-nitrophenylacetyl,o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl,isobutyryl, o-nitrocinnamoyl, picolinoyl, acylisothiocyanate,aminocaproyl, benzoyl and the like, and acyloxy includingmethoxycarbonyl, 9-fluoroenylmethyloxycarbonyl,2,2,2-trifluorethoxycarbonyl, 2-trimethylsilylethoxycarbonyl,vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (BOC),1,1-dimethylpropynyloxycarbonyl, benzyloxycarbonyl (CBZ),p-nitrobenzyloxyarbony, 2,4-dichlorobenzyloxycarbonyl, and the like.

The term "acid labile amine protecting group" means an amine protectinggroup as defined above which is readily removed by treatment with acidwhile remaining relatively stable to other reagents. A preferred acidlabile amine protecting group is tert-butoxycarbonyl (BOC).

The term "hydrogenation labile amine protecting group" means an amineprotecting group as defined above which is readily removed byhydrogenation while remaining relatively stable to other reagents. Apreferred hydrogenation labile amine protecting group isbenzyloxycarbonyl (CBZ).

The term "acid protecting group" means an easily removable group whichis known in the art to protect a carboxylic acid (--CO₂ H) group againstundesirable reaction during synthetic procedures and to be selectivelyremovable. The use of carboxylic acid protecting groups is well known inthe art and many such protecting groups are known, cf, for example, T.H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2ndedition, John Wiley & Sons, New York (1991), incorporated herein byreference. Examples of carboxylic acid protecting groups include esterssuch as methoxymethyl, methylthiomethyl, tetrahydropyranyl,benzyloxymethyl, substituted and unsubstituted phenacyl,2,2,2-trichloroethyl, tert-butyl, cinnamyl, substituted andunsubstituted benzyl, trimethylsilyl, and the like, and amides andhydrazides including N,N-dimethyl, 7-nitroindolyl, hydrazide,N-phenylhydrazide, and the like.

The term "hydrogenation labile acid protecting group" means an acidprotecting group as defined above which is readily removed byhydrogenation while remaining relatively stable to other reagents. Apreferred hydrogenation labile acid protecting group is benzyl.

"Aryl" mean sa phenyl or naphthyl group.

"Substituted aryl" means a phenyl or naphthyl group substituted by oneor more aryl group substitutents which may be the same or different,where "aryl group substituent" includes alkyl, alkenyl, alknyl, aryl,aralkyl, hydroxy, alkoxy, aryloxyl, aralkoxy, hydroxyalkyl, acyl,formyl, carboxyl, alkenoyl, aroyl, halo, nitro, triahalomethyl, cyano,alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino,aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarboamoyl, arylcarbamoyl,aralkylcarbamoyl, alkylsulfonyl, alkylsulfinyl, arysulfonyl,arylsulfinyl, aralkylsulfonyl, aralkylsulfinyl, or --NR_(a) R_(b) whereR_(a) and R_(b) are independently hydrogen, alkyl, aryl, or aralkyl.

"Aralkyl" means an alkyl group substituted by an aryl radical. Preferredaralkyl groups include benzyl, naphth-1-yl-methyl naphth-2-ylmethyl, andphenethyl.

"Substituted aralkyl" means an aralkyl group substituted on the arylportion by one or more aryl group substituents.

"Heterocyclyl" means about a 4- to about a 15-membered monocyclic ormulticyclic ring system in which one or more of the atoms in the ring isan element other than carbon, for example nitrogen, oxygen, or sulfur.Preferred heterocyclyl groups include pyridyl, pyrimidyl, andpyrrolidyl.

"Substituted heterocyclyl" means a heterocyclyl group substituted by oneor more aryl group substituents.

"Heterocyclyalkyl" and "substituted heterocyclyalkyl " means an alkylgroup which is substituted by a heterocyclyl and substitutedheterocyclyl group, respectively.

"Hygroscopicity" means sorption, implying an acquired amount or state ofwater sufficient to affect the physical or chemical properties of thesubstance (Eds. J. Swarbrick and J. C. Boylan, Encyclopedia ofPharmaceutical Technology, Vol. 10, p. 33).

Preferred Embodiments

A preferred compound prepared according to the present invention isdescribed by formula II wherein E² is H, alkyl, hydroxymethyl,1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl,2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl,cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl,substituted aryl, aralkyl, substituted aralkyl, heterocyclyl,substituted heterocyclyl, heterocyclylalkyl, substitutedheterocyclylalkyl, or E¹ and E² taken together with the nitrogen andcarbon atoms through which E¹ and E² are linked form a 4-, 5-, 6-, or7-membered azacycloalkane ring, provided that heterocyclylalkyl is otherthan indol-3-ylmethyl.

A more preferred compound prepared according to the present invention isdescribed by formula II wherein E² is H, alkyl, hydroxymethyl,1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl,2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl,cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkyalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, or E¹ and E² taken together with thenitrogen and carbon atoms through which E¹ and E² are linked form a 4-,5-, 6-, or 7-membered azacycloalkane ring.

A still more preferred compound prepared according to the presentinvention is described by formula II wherein E² is H, alkyl,hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl,carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl,cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, orE¹ and E² taken together with the nitrogen and carbon atoms throughwhich E¹ and E² are linked form a 4-, 5-, 6-, or 7-memberedazacycloalkane ring.

A further preferred compound prepared according to the present inventionis described by formula II wherein B is alkyl, cycloalkyl,cycloalkylalkyl, alkylcycloalkyl, or alkylcycloalkylalkyl.

A special embodiment prepared according to the present invention isdescribed by formula IIa ##STR9## wherein

B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl,alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl;

J is H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl,alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl or substitutedaralkyl;

L is OR¹ or NR¹ R² ;

R¹ and R² are independently H, alkyl, cycloalkyl, cycloalkylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl oralkylaralkyl;

m is 1 to 5;

n is 2 to 6; and

p is 1 or 2.

A more preferred special embodiment prepared according to the presentinvention is described by formula IIa wherein

B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl oralkylcycloalkylalkyl;

J is H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl oralkylcycloalkylalkyl;

m is 3; and

n is 3 or 4.

A further preferred special embodiment prepared according to the presentinvention is described by formula IIa wherein

B is alkyl;

J is alkyl, cycloalkyl or cycloalkylalkyl;

R¹ and R² are independently H, alkyl, cycloalkyl, cycloalkylalkyl,alkylcycloalkyl or alkylcycloalkylalkyl;

m is 3;

n is 3 or 4; and

p is 1.

A yet further preferred special embodiment prepared according to thepresent invention is described by formula IIa which isN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide.

Another embodiment according to the invention is the formation of astable non-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide. According to the invention, this form of the compound is capableof development as a stable formulation of the compound. The stablenon-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide also has a high melting point and shows no tendency to absorbwater. The stable form also exhibits unique and unexpected stabilityagainst humidities and temperatures well in excess of those normallyencountered upon shipping, dosage form manufacturing, or long termshippage or storage. These properties also facilitate dosage formmanufacturing. The conversion to the stable form also does not result inthe loss of material or its purity, and does not adversely affect itsparticle properties.

It is to be understood that the present invention is intended to coverall combinations of preferred compounds, preferred embodiments andspecial embodiments as defined herein.

A compound of the present invention is useful in the form of the freebase or acid, zwitterionic salt thereof or in the form of apharmaceutically acceptable salt thereof. All forms are within the scopeof the invention.

Where a compound of the present invention is substituted with a basicmoiety, an acid addition salt is formed and is simply a more convenientform for use; and in practice, use of the salt form inherently amountsto use of the free base form. The acid which can be used to prepare anacid addition salt includes preferably that which produces, whencombined with the free base, a pharmaceutically acceptable salt, thatis, a salt whose anion is non-toxic to a patient in the pharmaceuticaldoses of the salt, so that the beneficial inhibitory effects on plateletaggregation and thrombus formulation inherent in the free base are notvitiated by side effects ascribable to the anion. Althoughpharmaceutically acceptable salts of said basic compounds are preferred,all acid addition salts are useful as sources of the free base form evenif the particular salt, per se, is desired only as an intermediateproduct as, for example, when the salt is formed only for purposes ofpurification, and identification, or when it is used as intermediate inpreparing a pharmaceutically acceptable salt by ion exchange procedures.Pharmaceutically acceptable salts within the scope of the invention arethose derived from the following acids: mineral acids such ashyrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; andorganic acids such as acetic acid, citric acid, lactic acid, tartaricacid, malonic acid, methanesufonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,quinic acid, and the like. The corresponding acid addition saltscomprise the following: hydrohalides, e.g., hydrochloride andhydrobromide, sulfate, phosphate, nitrate, sulfamate, acetate, citrate,lactate, tartarate, malonate, oxalate, salicylate, propionate,succinate, fumarate, maleate, methylene-bis-β-hydroxynaphthoates,gentisates, mesylates, isethionates anddi-p-toluoyltartratesmethanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate,respectively.

According to a further feature of the invention, acid addition salts ofthe compounds of this invention are prepared by reaction of the freebase with the appropriate acid, by the application or adaptation ofknown methods. For example, the acid addition salts of the compounds ofthis invention are prepared either by dissolving the free base inaqueous or aqueous-alcohol solution or other suitable solventscontaining the appropriate acid and isolating the salt by evaporatingthe solution, or by reacting the free base and acid in an organicsolvent, in which case the salt separates directly or can be obtained byconcentration of the solution.

The acid addition salts of the compounds of this invention can beregenerated from the salts by the application or adaptation of knownmethods. For example, parent compounds of the invention can beregenerated from their acid addition salts by treatment with an alkali,e.g., aqueous sodium bicarbonate solution or aqueous ammonia solution.

Where the compound of the invention is substituted with an acid moiety,base addition salts may be formed and are simply a more convenient formfor use; and in practice, use of the salt form inherently amounts to useof the free acid form. The bases which can be used to prepare the baseaddition salts include preferably those which produce, when combinedwith the free acid, pharmaceutically acceptable salts, that is, saltswhose cations are non-toxic to the animal organism in pharmaceuticaldoses of the salts, so that the beneficial inhibitory effects onplatelet aggregation and thrombus formulation inherent in the free acidare not vitiated by side effects ascribable to the cations.Pharmaceutically acceptable salts, including for example alkali andalkaline earth metal salts, within the scope of the invention are thosederived from the following bases: sodium hydride, sodium hydroxide,potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithiumhydroxide, magnesium hydroxide, zinc hydroxide, ammonia,ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine,choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine,procaine, N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and thelike.

Metal salts of compounds of the present invention may be obtained bycontacting a hydride, hydroxide, carbonate or similar reactive compoundof the chosen metal in an aqueous or organic solvent with the free acidform of the compound. The aqueous solvent employed may be water or itmay be a mixture of water with an organic solvent, preferably an alcoholsuch as methanol or ethanol, a ketone such as acetone, an aliphaticether such as tetrahydrofuran, or an ester such as ethyl acetate. Suchreactions are normally conducted at ambient temperature but they may, ifdesired, be conducted with heating.

Amine salts of compounds of the present invention may be obtained bycontacting an amine in an aqueous or organic solvent with the free acidform of the compound. Suitable aqueous solvents include water andmixtures of water with alcohols such as methanol or ethanol, ethers suchas tetrahydrofuran, nitriles, such as acetonitrile, or ketones such asacetone. Amino acid salts may be similarly prepared.

The base addition salts of the compounds of this invention can beregenerated from the salts by the application or adaptation of knownmethods. For example, parent compounds of the invention can beregenerated from their base addition salts by treatment with an acid,e.g., hydrochloric acid.

As well as being useful in themselves as active compounds, salts ofcompounds of the invention are useful for the purposes of purificationof the compounds, for example by exploitation of the solubilitydifferences between the salts and the parent compounds, side productsand/or starting materials by techniques well known to those skilled inthe art.

Compounds of the present invention may contain asymmetric centers. Theseasymmetric centers may independently be in either the R or Sconfiguration. It will also be apparent to those skilled in the art thatcertain compounds of formula I may exhibit geometrical isomerism.Geometrical isomers include the cis and trans forms of compounds of theinvention having alkenyl moieties. The present invention comprises theindividual geometrical isomers and stereoisomers and mixtures thereof.

Such isomers can be separated from their mixtures, by the application oradaptation of known methods, for example chromatographic techniques andrecrystallization techniques, or they are separately prepared from theappropriate isomers of their intermediates, for example by theapplication or adaptation of methods described herein.

U.S. patent application Ser. Nos. 08/138,820 and 08/476,750 which areincorporated herein by reference describe methods for preparing anamorphous compound of formula II, and in particular, an amorphouscompound of formula I.

A novel process according to the invention for preparing a compound offormula II, and in particular, a crystalline compound of formula Iaccording to this invention is described by the synthesis shown inScheme II, wherein B, E¹, E², G, R, m, n and p are as defined above, andP¹ is a hydrogenation labile acid protecting group such as benzyl, P² isan acid labile amine protecting group such as t-butoxycarbonyl (BOC),and P³ is a hydrogenation labile amine protecting group such asbenzyloxycarbonyl (CBZ). ##STR10##

During the preparation of compounds of formula II or intermediatesthereto, it may also be desirable or necessary to prevent cross-reactionbetween chemically active substituents on these present on naturallyoccuring or pseudo amino acids. The substituents may be protected bystandard blocking groups which may subsequently be removed or retained,as required, by known methods to afford the desired products orintermediates (see, for example, Green, "Protective Groups in OrganicSynthesis", Wiley, New York, 1981). Selective protection or deprotectionmay also be necessary or desirable to allow conversion or removal ofexisting substituents, or to allow subsequent reaction to affort thefinal desired product.

The process of Scheme II is exemplified by the preparation of thecompound of formula II, however it should be understood that a compoundof formula I is prepared using the appropriate starting materials. Inthe preparation of the compound of formula I according to Scheme II, Bis ethyl, E¹ is H, E² is cyclohexylmethyl, G is NH₂, R is H, m is 3, nis 3, p is 1, P¹ is benzyl, P² is BOC, and P³ is a CBZ.

An alternative process according to the invention for preparing acompound of formula I is the same as that in Scheme II except that thecompound of the formula III, wherein P³ is as defined above, is used inplace ##STR11## of the compound of formula IV, wherein R is H, m is 3, nis 3, p is 1, and P³ is a ##STR12## CBZ to yield a intermediate offormula V, wherein B is ethyl, E¹ is H, E² is ##STR13##cyclohexylmethyl, G is NH₂, p is 1, P¹ is benzyl, and P³ is a CBZ.

Scheme II demonstrates a five step method of preparing a compoundaccording to the invention starting with the formation of a centraldipeptide intermediate according to the invention of the formula VI,wherein B, p, P² and ##STR14## P¹ are as defined above. In the case ofthe preparation of the compound of formula I, the central dipeptideintermediate according to the invention isBOC-N(Et)Gly-(L)-Asp(OBzl)-OH. The central dipeptide intermediate isprepared without protection of the free carboxylic acid moiety.

In step 2 of Scheme II, the coupling to form the central dipeptide maybe effected in either dichloromethane or mixtures of ethyl acetate--withor without DMF as cosolvent--and organic bases such as NMM, and may bedone at about room temperature to about 40° C. Activation of the aminoacid or pseudo amino acid of the following formula for coupling may beeffected using ##STR15## non-isolated active esters with p-nitrophenol,pentafluoro-phenol, and N-hydroxy-succinimide via the action ofdicyclohexylcarbodiimide. Coupling times range from about 1 to about 20hours, depending upon the amino acids or pseudo amino to be coupled,activating agent, solvent, and temperature. The central dipeptideproduct of step 1 does not have to be isolated. The step 1 reactionmixture is typically washed with water or dilute aqueous acid (eg. aq.HCl), and then used directly without drying in step 2. In the instancewhen a phenol-based active esters is used, the central dipeptide productis extracted into alkaline water from the reaction mixture, thenre-extracted from the acidified aqueous solution back into an organicsolvent; and the solution is reacted directly as in step 2.

The dipeptide intermediate of formula VI is used to prepare a tripeptideintermediate according to the invention of formula VII, wherein B, E, F,G, p and ##STR16## P¹ are as defined above, and P^(2') is P² or TFA•H--,Where P^(2') is TFA•H--. The "•" symbol represents dissociation of theTFA to form F₃ CCO₂ ⁻ and H⁺, wherein the H⁺ protonates the terminalamine in the compound of formula VII, i.e., yielding the TFA salt offormula VIIa. In the case of the preparation of the compound of##STR17## formula I, the tripeptide intermediate according to theinvention is P^(2') -N(Et)Gly-(L)-Asp(Obzl)-(L)-Cha-NH₂.

In step 2, the coupling of an amino acid or pseudo amino acid to thecentral dipeptide may be effected in either dichloromethane or inmixtures of ethyl acetate and DMF or THF, and at about or below roomtemperature. Activation of the central dipeptide of the followingformula for coupling may be ##STR18## effected using non-isolated activeesters of pentafluorophenol or N-hydroxy-succinimide via the action ofdicyclohexylcarbodiimide. Activation may also be effected usingisopropyl chloroformate. Reaction times vary with the amino acids orpseudo amino acids to be coupled, activating agent, solvent, andtemperature, and range from about 1 to about 20 hours. The tripeptideproduct does not have to be isolated. When the tripepetide intermediateis not isolated, the reaction mixture is washed with aqueous organicbase such aq. N-methyl morpholine and aqueous acid such as aq. HCl andis reacted "as is" via the method of Step 3 after the aqueous washingsand without drying.

In Step 3 of Scheme II, the removal of the protecting group such as BOCfrom the tripeptide product of Step 2 may be effected using a solutionof trifluoroacetic acid in dichloromethane, or using a mixture of HBr inacetic acid and ethyl acetate. The reaction may be run at about roomtemperature, and requires about 1 hour (HBR method) and about 2 hours(TFA method). The acid salt product of the tripeptide is isolated byfiltration as a crystalline solid either directly from the reactionmixture (HBr method), or after partial solvent removal by distillationand addition of a non-polar solvent to the pot residue.

A further process according to the invention is described as a singleconcatenated process to rapidly and simply prepareTFA•H-N(Et)-Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ from BOC-N(Et)Gly-OH, whichprocess is a one-pot reaction encompassing the first two coupling stepsin Scheme II and the treatment with TFA. TheTFA•HN(Et)-Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ is obtained singularly as itcrystallizes directly from the concatenated reaction solution. Theconcatenated process avoids the corresponding three discreet reactionsin Scheme II and solves the problem to establish a simple, time- andcost-effective synthesis which is useful in a manufacturing environment.

Scheme II shows the construction of a polypeptide in reverse order,beginning with an N-protected amino acid and then adding successively tothe carboxyl terminus, as opposed to the conventional order, in which apolypeptide is constructed by successive amidations at the amineterminus of a protected C-terminus amino acid. This reverse syntheticmethod according to the invention requires nitrogen protection of onlythe first amino acid, enabling the use from that point onward ofsuccessive amino acids having no protection at either the amine or acidterminus (side chain functional groups excepted). The reverse syntheticmethod also streamlines production of a compound of formula II, and inparticular a compound of formula I, by enabling the use of flow typemanufacturing technology as opposed to batch type normally required forsolution phase peptide chemistry. The new approach cuts production costby removing the requirement to purchase amino acids protected at theamino terminus. No special equipment, reagents, or analyticalmethodology are required.

Another process according to the invention is the formation of stablenon-hygroscopic crystallineN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide reproducibly obtained by a novel solid state conversion fromhygroscopic crystallineN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide prepared by the method as described in Scheme II and the notedalternative reaction steps.

The hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide is physically unstable, and is converted upon exposure toconditions of humidity and temperature to the highly stable,non-hygroscopic crystalline formN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide.

The general conditions according to the invention for the conversionfrom the hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide to the highly stable, non-hygroscopic crystalline formN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide have been effected under static and dynamic conditions.

The static procedure according to the invention is described as a staticconversion because it involves exposing the hygroscopic crystalline formofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-.beta.-cyclohexylalanineamide placed in a non-moving vessel such as in vials or trays to certainconditions of temperature and humidity in a controlled environmentalchamber. This "static" conversion is performed at temperatures andrelative humidities ranging from about 20° C. to about 80° C., morepreferably at about 40° C. to about 80° C., and at about 40% to about100% RH, preferably about 65 to about 80% RH.

The dynamic procedure according to the invention is described as adynamic conversion because it involves exposing the hygroscopiccrystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-.beta.-cyclohexylalanineamide under incubation at the humidity and temperature levels as in thestatic model, but also under a means of agitation including tumbling ofthe hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide in a rotary evaporation flask or in a cylindrical vessel (in ahumidity oven) with propeller agitation.

The following Examples are illustrative of the invention and are notintended to limit the scope.

Unless otherwise indicated, reported mass spectral analysis data are LowResolution Fast Atom Bombardment performed on a VG 70SE with"calculated" values being (M+H)⁺. Nuclear magnetic resonance (NMR)spectral data is obtained on a Bruker ACF 300, in D₂ O. Flashchromatography is done on silica gel. High performance liquidchromatography (HPLC) is done on a C-18 Reverse Phase columns ofparticle size ranging from 8-15μ.

Unless otherwise indicated, reported x-ray powder diffraction graphs areobtained using a Siemens D5000 diffractometer with a Cu radiation source(1.8 kW, 45 kV and 40 mA) to scan powder samples. The samples are milledprior to measurement to eliminate particle size effect on the peakintensities. Approximately 60 mg of the sample are loaded into a 1.5×1cm sample holder and scanned in the range 3-40° 2 theta (2θ) with stepsize of 0.04° and the total exposure of 1 second per step.

EXAMPLE 1 Preparation of BOC-N(Et)Gly-(L)-Asp(OBzl)-OH (Step 1 of SchemeII)

Into a 1 L 3-neck round bottom flask are charged 51 g (0.25 mole) ofBOC-N(Et)Gly-(L)-Asp(OBzl)-OH, 35 g (0.25 mole) of PNP, 400 mL of EtOAc,and 100 mL of DMF. The mixture is stirred to form a solution and cooledto 4-6° C. A solution of 51.5 g (0.25 mole) of DCC in 125 mL EtOAc isadded dropwise over a period of 10 minutes, while maintaining thetemperature from about 5° C. to about 8° C. After all DCC is added, thecooling bath is removed and the mixture is allowed to stir for 1.5 hoursas it warmed to room temperature (20-22° C.). A solid precipitate, DCUforms during this period. Completeness of formation of the PNP ester isdetermined by analytical HPLC (disappearance of BOC-N(Et)Gly-OH). Thereaction mixture is filtered and the DCU residue is washed with 2-50 mLportion of EtOAc and the washes added to the filtrate. The DCU isdiscarded.

To the stirred, filtered solution is added 67 g (0.3 mole) of H₂N-(L)-Asp(OBzl)-OH as a slurry in 150 mL (138 g, 1.36 mole) of NMM. Themixture is heated to 38-40° C. and maintained at that temperature for 41hours, the point at which an analytical HPLC indicates completeconsumption of BOC-N(Et)Gly-OPNP. The reaction mixture is cooled to 25°C. and unreacted H₂ N-(L)-Asp(OBzl)-OH is filtered off. The solution iscooled and refiltered to afford an additional 1.2 g (21.7 g recovered;11.2 g represents the 20% excess added and 10.5 g (0.047 mole)represents unreacted material).

The filtered solution is extracted in a 2 L Squibb funnel with oneportion of 500 mL deionized water, followed with 2-250 mL portions. Thecombined aq. solution is extracted with 3-300 mL portions of 1:1MTBE/EtOAc to remove residual PNP (HPLC anal. shows only a traceremaining), then is cooled to 5° C. and acidified from pH 8.9 to pH 1.79by dropwise addition of 150 mL concentrated HCl. The acidified aqueoussolution is extracted with 2-200 mL portions of EtOAc. HPLC analysis ofthe aqueous shows no residual desired product. The EtOAc extracts arecombined, dried over MgSO₄, filtered, and concentrated by rotaryevaporation at 35° C. The resulting pale orange oil is pumped at 35° C.to maximize removal of residual solvent to afford 85.68 g ofBOC-N(Et)Gly-(L)-Asp(OBzl)-OH as an oil (21.3 mmol, 85.5% yield,uncorrected for residual solvent).

Characterization:

NMR (250 MHz): 7.3 ppm (s), 5.1 ppm (s), 3.3 pm (dq), 3.0 (dq), 1.4 ppm(s), 1.1 (t) MS: M=408; M+1_(obsvd) =409 HPLC: 90.79A% (3.87A%p-nitrophenol, uncorrected for e) Elemental analysis: C₂₀ H₂₈ N₂ O₇ : H,N; C_(fd) 57.54, C_(cal). 58.81

EXAMPLE 2 Preparation of BOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (Step 2of Scheme II)

Method A: Isopropyl Chloroformate Method

One equivalent BOC-N(Et)Gly-(L)-Asp(OBzl)-OH is dissolved into EtOAc,(6-8 volumes; 1:6.5 Wt:vol) and maintained at a temperature between-15-0° C. NMM (1 equivalent) is added while maintaining the temperaturefrom about -15° C. to about 0° C. Isopropyl chloroformate (1-1.1equivalents) is added into the protected dipeptide solution at atemperature between -15-0° C. The reaction is maintained at atemperature between about -15° C. to about 0° C. for two to fiveminutes. A solution of H₂ N-(L)-Cha-NH₂, (1 equivalent), in THF, (10volumes; 1:10 Wt:vol) is added to the cooled dipeptide solutionmaintaining temperature at about -15° C. to about 0° C. The reaction ismonitored with in-process control (HPLC) samples obtained at 15 minutes,1 hour, and 2 hours to evaluate reaction completion. (The reaction iscomplete when the amount of observed dipeptide is less than 10% by areaby HPLC analysis).

The BOC-tripeptide product precipitates directly from the reactionsolution and is filtered from the reaction mixture, washed with EtOAc(2X, 1 volume; Wt:vol), and dried under vacuum. Typical yields are >60%,with purities >90A %; <1A % of the aspartic acid-epimeric diastereomerhas been typically observed.

An EtOAc reslurry provides final yields of ˜60% ofBOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ and improves purity to >95A %while reducing the diastereoisomer to <0.5%.

As a specific example of the isopropyl chloroformate method, when thegeneral procedure of Example a is followed and 4.55 g (8.1 mmole) ofBOC-N(Et)Gly-(L)-Asp(OBzl)-OH is used, then the amount ofBOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ prepared is 3.26 g (97.9A % pure,0.3A % diastereomer), a 70% theoretical yield.

Method B: Pentafluoro-Phenol-DCC Complex Method

Pentafluoro phenol (PFP, 2.9 equivalents) and DCC (1 equivalent, aredissolved into EtOAc, (5 volumes; 1:5 Wt:vol) at room temperature andcooled to a temperature between -15-0° C. One equivalentBOC-N(Et)Gly-(L)-Asp(OBzl)-OH is dissolved into EtOAc, (6 volumes; 1:6Wt:vol) and mixed with one equivalent of H₂ N-(L)-Cha-NH₂ which ispreviously dissolved into DMF, (10 volumes; 1:10 Wt:vol). Thedipeptide/H₂ N-(L)-Cha-NH₂ solution is added dropwise into the solutionof PFP and DCC, maintaining temperature between -15-0° C. The reactionis maintained at a temperature between 15-22° C. for five to sixteenhours with in-process control (HPLC) samples obtained at 1, 2, 3, 4, and16 hours to evaluate reaction completion. (The reaction is completedwhen the amount of observed dipeptide is less than 2% by area by HPLCanalysis).

The reaction mixture is filtered and the filter cake (DCU) washed withEtOAc, (2×0.5 volumes; Wt:vol). The filtrate is treated with water, (10volumes; 1:10 Wt:vol) and the water layer removed. The EtOAc layer iswashed with water, (1×, 5 volumes: 1:5 Wt:vol). The EtOAc layer iscooled to precipitate out the product, which is filtered and washed withEtOAc, (2×0.4 volumes; 1:0.4 Wt:vol). Isolated molar yields are >60%with typical purities of >90A %, with 1-4A % of the asparticacid-epimeric diastereomer.

An EtOAc reslurry provides final yields of ˜60% ofBOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ and improves purity to >99A %while reducing the diastereoisomer to <0.5%.

As a specific example of the pentafluoro-phenol-DCC complex method, whenthe general procedure of Method B is followed and 10 g (24.5 mmole) ofBOC-N(Et)Gly-(L)-Asp(OBzl)-OH is used, then the amount ofBOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ prepared is 8.15 g (99A % pure,0.49A % diastereomer), a 59% theoretical yield.

Method C: Hydroxybenzotriazole(HOBT)/2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU) Method

One equivalent BOC-N(Et)Gly-(L)-Asp(OBzl)-OH is dissolved in DMF (9-10volumes; 1:10 Wt:wt) and maintained at ambient temperature. To thissolution is added H₂ N-(L)-Cha-NH₂ (1 equivalent) andhydroxybenzotriazole (HOBT, 1 equivalent). The resulting solution iscooled to about 0° C. to about 10° C., and NMM (1-1.1 equivalents) isadded. The coupling reagent, TBTU, (1-1.1 equivalents) is dissolved intoDMF, (4-5 volumes; 1:5 Wt:wt) and is added to the protected dipeptidesolution at a temperature of 0° C. to about 10° C. This solution isstirred at about 10° C. to about 25° C. for about 3 hours, until HPLCanalysis indicated completion of the reaction (less than 2% startingmaterial by area). The reaction mixture is added to a stirred mixture of5% aqueous sodium chloride (about 4 volumes vs. reaction volume) andEtOAc (about 2 volumes vs. reaction volume). The phases are separated,and the aqueous phase is extracted with an additional portion of EtOAc(about 1.5 volumes vs. reaction volume). The organic phases are combinedand washed sequentially with 0.5 N aqueous citric acid (about 0.6-0.7volumes vs. organic phase volume), 10% aqueous sodium bicarbonate(twice, with about 0.6-0.7 volumes vs. organic phase volume each) and25% aqueous sodium chloride (about 0.3-0.4 volumes vs. organic phasevolume). The resulting organic phase is concentrated to about 1/4 to 1/2volume under reduced pressure at about 30-50° C., and to this warmsolution is added an equal volume of heptane. The mixture is stirred andallowed to cool to about 0° C. to about 20° C. to precipitate thedesired tripeptide. This solid is filtered, washed with a mixture ofEtOAc and heptane, and dried. A typical yield is >60%, with typicalpurities of >95.7A % and levels of the aspartic acid-epimericdiastereomer at <2A %.

As a specific example of HOBT/TBTU Method, when the general procedure isfollowed, 10 g (24.5 mmole) of BOC-N(Et)Gly-(L)-Asp(OBzl)-OH is used,then 9.3 g of BOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ is prepared (96.1A% pure, 1.77A % diastereomer at Asp), a 67.7% theoretical yield.

Mass Spec: M_(calc) 560.7; M+1_(obsvd) 561 mp 182.17(DSC) ¹ H NMR (δ vsTMS, D6 DMSO): 0.89 m (1H); 0.94, m (1H); 1.0, dt (2H); 1.15, m (2H):1.06-1.3, m (4H); 1.36, d (9H); 1.4-1.74, m (6H); 2.65, m (1H); 2.85, m(1H); 3.18, m (2H); 3.75, d (2H); 4.2, s (1H); 4.66, d (1H); 5.08, s(2H); 7.02, s (1H); 7.18, d (1H); 7.36, s (5H); 7.88, dd (1H); 8.24, dd(1H).

EXAMPLE 3 Preparation of TFA-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (Step 3of Scheme II)

BOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ is dissolved in dichloromethane(˜1:12 wt/wt), and to that solution is added TFA at ambient temperature.This is then stirred until HPLC indicates complete reaction (3-5 hours).The solution is concentrated to about 1/2 volume at 40-45° C. To thiswarm solution is added MTBE (˜1:10 wt/wt vs.BOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂) while maintaining thetemperature >40° C. The mixture is slowly cooled to about 5° C. andstirred for 1 hour to ensure complete crystallization. The resultingsolids are filtered, and washed with chilled MTBE. The solids are driedunder reduced pressure and analyzed for content ofTFA.N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (HPLC wt/wt assay). Yield isgenerally nearly quantitative, purity >95A %.

Mass Spec: M_(calc). 460 (free base); M+1_(obsvd) : 461 Elementalanalysis: C₂₆ H₃₇ N₄ O₇ F₃ H,N, F, C 54.35, fd., 53.82 ¹ H NMR (δ vsTMS, D⁶ DMSO): 0.9, m (2H); 1.15, t (6H); 1.5, m (1H); 1.5-1.8, m (6H);2.65 dd (1H); 2.9 m (3H); 3.7, s, (2H); 3.9, m (2H); 4.2, m (1H); 4.75,m (1H); 5.1, s (2H); 7.0, s (1H); 7.15, s (1H); 7.2, s (5H); 8.13, d(1H); 8.7-8.8, m (3H). ¹³ C NMR (salient signals, δ vs TMS, D6 DMSO):10.76, 25.49, 25.68, 25.96, 31.66, 33.07, 33.36, 36.25, 38.59, 41.88,47.02, 49.40, 50.47, 65.71, 127.81-128.34, 135.82, 165.10, 169.34,173.79

Specific examples of the deprotection are shown in Table A.

                  TABLE A                                                         ______________________________________                                        Lab    Reaction scale amount                                                                          Yield and A% purity                                   ______________________________________                                        Example                                                                              (BOC--N(Et)Gly--(L)--Asp                                                                       (TFA · N(Et)Gly--(L)--Asp                           (OBzI)--(L)--Cha--NH.sub.2.)                                                                   (OBzI)--(L)--Cha--NH.sub.2)                           Example 1                                                                            7.5 g (13.3 mmole)                                                                             7.4 g (12.9 mmole) 97%                                                        yield; 98.8A% pure                                    Example 2                                                                            6.53 g (11/6 mmole)                                                                            6.4 g (11.1 mmole) 96%                                                        yield; 98.47A% pure                                   ______________________________________                                    

EXAMPLE 4 Preparation of CBZ-PipBu-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂(Step 4 of Scheme II)

A suspension of ˜equimolar amounts ofTFA.N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂, CBZ-PipBu, and TBTU in EtOAc,DMF, and water (100:8:4 v/v, ˜11:1 total v/wt vs.TFA-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂) are prepared. This suspension iscooled to 0-10° C. and about 3-4 equivalents of NMM is added. Thismixture is allowed to warm to room temperature and stirred until HPLCindicates complete reaction (1-3 hours; solution occurs during thistime). Water is added (2-3 X original amount of water added) and thephases allowed to separate. The aqueous phase is reserved and theorganic phase washed with two more portions of water. These combinedaqueous washes are back-extracted with EtOAc, and the combined organicphases washed with 25% aqueous sodium chloride. The organic phase isconcentrated under reduced pressure to ˜1/2 volume, and MTBE (˜1/2 v:vvs. solution volume) added. This mixture is allowed to crystallize(several hours), and the solids are collect by filtration, rinsing witha chilled mixture of EtOAc and MTBE. The solids are dried under reducedpressure. The content of CBZ-PipBu-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ isanalyzed by HPLC wt/wt assay. Yield is generally >80%, purity >95A %

As a specific example of the above preparation, when the generalprocedure of Step 4 is followed, 7.25 g ofTFA.N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ provides 7.9 g ofCBZ-PipBu-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (>99A % pure, 0.08A %diastereomer at Asp), an 84% theoretical yield.

Elemental Analysis C₄₁ H₅₇ N₅ O₈ :H, N, C, 65.84, fd., 65.38 Mas Spec:M_(calc) 757; M+1_(obsvd) 748 mp 101.6 (DSC) ¹ H NMR (δ vs TMS, CDCl₃):0.88 m (1H); 0.98, m (1H); 1.13 (2H); 1.23, m (6H); 1.4, m (1H);1.62-1.76, m (8H); 1.86, qd (1H); 2.35, t (1H); 2.74, dd (2H); 3.25, dd(1H); 3.47, q (2H); 3.7, d (1H); 3.84, d (1H); 4.15, ds (2H); 4.5, qd(1H); 4.68, dt (1H); 5.07, d (1H); 5.14 bd (2H); 5.16, d (1H);7.28-7.39, m (10H); 7.57, dd (1H) ¹³ C NMR (δ vs TMS, CDCl₃): [salientpeaks] 66.93 (both benzyl carbons), 127.78-128.64 (both phenyl rings),155.249, 170.00, 170.24, 171.69, 174.27, 175.21 (all carbonyl carbons)

EXAMPLE 5 Preparation of Hygroscopic Crystalline Form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (Step 5 of Scheme II)

A mixture of CBZ-PipBu-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂, ammoniumformate, and 10% Pd/C in 20:1 alcohol/water (10:1 v/wt vs.CBZ-PipBu-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂) is prepared. This mixtureis heated to 40-50° C., and stirred until HPLC indicates completereaction (1-2 hours). The mixture is cooled to room temperature andfiltered to remove the catalyst. The resulting solution is heated to40-50° C. and acetone added (˜equal volume vs. filtered solution),allowing the solution to cool to 35-40° C. Seeds ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide are added to the mixture and hygroscopic form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide crystallizes therefrom while cooling to room temperature (severalhours). The solids are collected by filtration under a blanket ofnitrogen, rinsing with acetone. The solids are dried under reducedpressure and analyzed for content of the hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl](L)-β-cyclohexylalanineamide (HPLC wt/wt assay). Yield is generally >85%, purity >95A %.

As a specific example of the above preparation, when the generalprocedure of Step 5 is followed, 5 g ofCBZ-PipBu-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ provides 3.1 g of ahygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide as a white solid (99.6A % pure), a stoichiometric yield of 89.4%.

Other compounds prepared according to the above Examples 1-5, but usingthe appropriate starting materials, include the following:

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]valine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-D-valine,

N-[N-[N-(3-(piperidin-4-yl)propanoyl)-N-ethylglycyl]aspartyl]valine,

N-[N-[N-(5-(piperidin-4-yl)pentanoyl)-N-ethylglycyl]aspartyl]valine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-α-cyclohexylglycine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]norleucine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-α-(2,2-dimethyl)prop-3-ylglycine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-β-decahydronaphth-1-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-α-(2-cyclohexylethyl)glycine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]phenylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-β-naphth-1-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-β-naphth-2-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-β-cyclohexylalanine, ethyl ester,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-L-β-cis-decahydronaphth-2-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-α-aminocyclohexanecarboxylicacid,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclohexyl-D-alanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-decahydronaphth-1-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclohexylalanineethyl amide,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclooctylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclohexylmethylalanineamide,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-adamant-1-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-(1,2,3,4)-tetrahydronaphth-5-ylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-(4-cyclohexyl)cyclohexylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cycloheptylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclooctylalanineamide,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-α-cyclohexylpropylglycine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclooctylmethylalanine,

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclopentylalanine,and

N-[N-[N-(4-(piperidin-4-yl)butanoyl)-N-ethylglycyl]aspartyl]-β-cyclohexylmethylalanineethyl ester, and

EXAMPLE 6 Preparation of 4-N-CBZ-piperidone

A mixture of 40 Kg N-benzyloxycarbonyloxy) succinimide and 26 Kg (175mol) 4-piperidone.Hcl.H₂ O in 38.8 Kg water and 88 Kg THF is stirred at15° C.±5° C. until dissolution is complete (˜15 minutes). NMM (22.8 Kg)is added to the agitated mixture (exothermic) while maintainingtemperature at or below 20° C. The batch is agitated at 20° C.±5° C. for2.5 hours, at which point HPLC indicated complete reaction. The mixtureis diluted with 115.2 Kg MTBE and 38.8 Kg of water and agitated at 20°C.±5° C. for 5 minutes. Agitation is stopped, the layers are allowed toseparate, and the aqueous (lower) layer is removed and discarded. Theorganic layer is washed with 2×129.6 Kg of water (agitate 5 minutes,separate phases, remove/discard aqueous [lower] phase ). The organiclayer is washed with 5.2 Kg of NaCl in 46.8 Kg of water (agitate 5minutes, separate phases, remove/discard aqueous [lower] layer). Theorganic layer is treated with 11.5 Kg MgSO₄, with agitation for 1 hour,then the mixture is filtered. The reactor is rinsed with 8 Kg MTBE(filtered, combined with main filtrate; total filtrate water content:0.52%). The mixture volume is reduced by half via distillation atreduced pressure at 30° C. Vacuum is broken to nitrogen and the residueis cooled to 20° C. (pot residue water content: 0.43%). The residue isdiluted with 57.6 Kg MTBE, then mixture volume is reduced again by halfvia distillation under vacuum at 30° C. Vacuum is released to nitrogenand the mixture is cooled to 20° C. (pot residue water content: 0.25%).This is repeated 5 additional times. The final pot residue is dilutedwith 28.8 Kg of MTBE and mixed for 5 minutes, then assayed for watercontent and content of 4-N-CBZ-piperidone (water: 0.05%; wt/wt assay4-N-CBZ-piperidone: 22.66 wt %, 35.35 kg, 155 mole, 88.6% stoich. yld.).

EXAMPLE 7 Preparation of PipBu

Under a N₂ purge and with agitation is prepared a solution of 53.5 Kg3-carboxypropyl triphenylphosphonium bromide in 230.1 Kg of1,2-dimethoxy-ethane. Potassium-tert-butoxide/THF (20 wt %, 141.8 Kg ofsoln.) is added over 35 minutes while maintaining the temperature at24-28° C. The mixture is stirred at this temperature for 0.5 hour, atwhich point HPLC indicates a complete reaction. The agitated mixture iscooled to 10° C.±2° C., then to the mixture is added 96.45 Kg (Titer:1.15 molar eq. vs.) of 4-CBZ-piperidone in MTBE over 40 minutes suchthat batch temperature remains at 12° C.±2° C. The mixture is agitatedat this temperature for 10 minutes, then is heated to 20° C.±2° C. andagitated at that temperature for 2 hours. To the agitated mixture isadded a solution of 22.5 Kg concentrated aq. HCl in 245.6 Kg of water soas to maintain the mixture at 20° C.±2° C.; the final pH is 0.5. Themixture is extracted, with agitation, with 214.0 Kg methyl-tert-butylether. Agitation is stopped, the phases are allowed to separate, and theaqueous layer (lower) is removed and discarded. The organic phase iswashed with 133.75 Kg of water (agitate 5 minutes, separate,remove/discard aqueous [lower] layer), then with 10.7 Kg 50% NaOH in126.8 Kg water (agitate 10 minutes, separate layers, removed/discardorganic [upper] layer). The aqueous layer is extracted with 2×123.05 KgEtOAc (agitate 5 minutes, separate layers, remove/discard organic[upper] layers). To the agitated aqueous layer is added 13.1 Kgconcentrated aq. HCl to a pH of 2.5-3.5 (final: 2.82), then the mixtureis extracted with 123.05 Kg EtOAc (agitate 5 minutes, separate layers,remove/discard aqueous [lower] layer). The EtOAc solution is washed with133.75 Kg water (agitate 5 minutes, separate layers, remove/discardaqueous [lower] layer), then is assayed (wt/wt) for content ofCBZ-PipBuen (total wt.: 194.86 kg, 17.05% CBZ-PipBuen [33.22 kg, 108mole], 87.9% stoich yld.).

The EtOAc solution of PipBuen, along with 6.6 Kg 5% Pd/C (50% water bywt.) is charged with agitation to a stainless steel pressure tank, thenthe mixture is heated to 55° C.±2° C. Potassium formate (38.2 Kg)dissolved in 66.4 Kg of water is added such that the reaction mixturetemperature remains at 55° C.±2° C. (˜30 minutes). The mixture isagitated at 55° C.±2° C. for 2 hours, at which time reaction wascomplete (HPLC). To the reactor is added 6.6 Kg celite and 33.2 Kgwater, the mixture agitated, then filtered. The reactor is rinsed with33.2 Kg of water (filtered, added to main filtrate). The filtrate isplaced in a new vessel, cooled to 20-25° C., the layers allowed toseparate, and the organic layer removed and discarded. The aqueous layeris acidified with 52.1 Kg of concentrated aq. HCl to pH 2-3 (final:2.82), then extracted with 4×129.5 Kg methylene chloride (agitate 5minutes, separate layers, remove/discard organic [lower] layers). Theaqueous phase is adjusted to pH 6.1 by addition, with agitation, of17.85 Kg 50% aq. NaOH. The mixture is filtered to afford a 224 Kgsolution containing 17.6 Kg (103 mole) of4-(3'-carboxypropyl)piperidine.

EXAMPLE 8 Preparation of CBZ-PipBu

The 224 Kg solution of 4-(3'-carboxypropyl)piperidine in aq., NaOH iscombined with 55.3 Kg THF and the mixture cools with agitation to 8°C.±2° C. NMM (20.9 Kg) is added while maintaining temperature at <10° C.After addition is complete, the temperature is adjusted to 8° C.±2° C.,then 25.7 Kg of 1-(benzyloxocarbonyl)succinimide dissolved in 49.8 Kg inTHF is added over 1 hour, while maintaining the temperature at <15° C.The reaction is complete (analytical HPLC) after 3 hours at 10-15° C.Concentrated aq. HCl (29.9 Kg) is added to adjust the pH to 2.5-3.5(final: 3.3), then 61.4 Kg MTBE is added and the mixture is agitated for5 minutes. Agitation is stopped, the layers allowed to separate, and theaqueous (lower) layer is separated (waste). The MTBE layer is washedwith three 83.1 Kg portions of water (10 minute, then 5 minute and 5minute agitation periods); the aqueous phase is allowed separate andremoved (waste) in each case. A solution of 8.3 Kg of 50% aq. NaOH in95.7 Kg water is added without agitation, then upon complete addition,the mixture is agitated for ˜5 minutes. Agitation is stopped, the phasesare allowed to separate, and the organic (upper) layer is separated anddiscarded. The aqueous layer is returned to the reactor and extractedwith 2×38.4 Kg of methyl-tert-butyl ether (agitated 5 minutes, layersseparated, organic [upper] layers removed/discarded). This operation isrepeated using 18.5 Kg methyl-tert-butyl ether. The aqueous layer,returned to the reactor, is acidified to pH 2.5-3.5 (final: 3.37) with9.9 Kg of concentrated aq. HCl. The mixture is extracted with 76.4 Kgmethyl-tert-butyl ether (agitate 5 minutes, separate layers, lower[aqueous] layer removed/discarded). The organic layer is washed (5minute agitation) with a solution of 1.1 Kg NaHCO₃ in 12.4 Kg of water(agitate 5 minutes, separate layers, aqueous layer [lower]removed/discarded), the with 41.5 Kg of water (agitate 5 minutes,separate layers, aqueous layer [lower] removed/discarded). The reactoris placed under reduced pressure and volatile solvents removed at 55° C.until distillate flow ceases. Toluene (32.4 Kg) is added, and themixture is distilled under atmospheric pressure until distillate flowstopped, while the batch temperature climbs to 90-95° C. The mixture isthen cooled to 30-35° C., heptane (56.85 Kg) is charged to the reactor(two phases), the mixture is heated to 90-95° C. (single phase), thenrecooled to 38-42° C. Seed crystals of CBX-PipBu are added, and theproduct crystallized from the mixture over a 1 hour period. The solid iscollected by filtration and washed with 19.35 Kg of 1:2 toluene/heptane,then with 33.4 Kg heptane. The filter cake is dried under vacuum at 40°C. (to 0.13% loss on drying analysis) to afford 22.4 Kg (72.96 mole, 42%stoich. yld from 4- piperidone) of CBZ-PipBu.

EXAMPLE 9 Preparation of CBZ-PipBuen

To a suspension of 82 g of 3-carboxypropyl triphenylphosphonium bromidein 407 mL 1,2-diethoxyethane at 14° C. is added over 25 minutes 220 g of20 wt % potassium tert-butoxide in tetrahydrofuran while maintaining thereaction mixture temperature at 24-28° C. The mixture is stirred for 1hour, cooled to 10° C., then a solution of 52.5 g of 4-N-CBZ-piperidonein 246 mL of tert-butyl methyl ether is added over 30 minutes, whilemaintaining cooling. After addition is complete, the mixture is stirredat 12° C. for 10 minutes, then warmed to 20° C. and stirred for anadditional 30 minutes. The reaction mixture is treated with 410 mL 1 Naq. HCl for 10 minutes, diluted with 328 mL of t-butyl methyl ether, andthen the phases are separated. The organic phase is washed with 205 mLof water, then 210 mL of 1 N aq. NaOH. The NaOH layer, which containsthe product, is collected separately, washed with three 189 g portionsof ethyl acetate, acidified to pH 3.48 with concentrated HCl, thenextracted with 189 mL of ethyl acetate. The ethyl acetate layer isseparated, washed with 211 mL of water, then dried for 30 minutes over10 g of MgSO₄, filtered, and concentrated in vacuo. The oily residue(50.7 g) is crystallized form toluene/heptane to afford a total of 29.46g (50.9% yield; ˜95A % pure) of CBZ-PipBuen.

Mass Spec: M_(calc). 303, M+1_(obsvd) 304 ¹ H NMR: (δ vs TMS, CDCl₃)2.2, t (2H); 2.25, t (2H); 2.35, m (4H); 3.45, m (4H), 5.15, s (2H);5.2, m (1H); 7.33, 2 (5H). ¹³ C NMR (δ vs TMS, CDCl₃) 22.43, 28.2,34.26, 35.66, 44.88, 45.74, 67.20, 122.02, 127.83, 127.95, 128.45,128.69, 128.90, 136.17, 136.72, 155.34, 178.39

EXAMPLE 10 Preparation of CBZ-PipBuen-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂(Alternate Step 4 of Scheme II)

CBZ-PipBuen (70 g, 0.23 mole) and DMF (230 mL) are added to a 1 Ljacketed flask and stirred with cooling to 0° C., then TBTU (74.9 g,0.23 mole) is added add at once. The temperature is maintained at 0° C.and the addition of DIPEA (61.9 g, 0.61 mole) is started. After 45 min.,TFA-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (138.7 g, 0.24 mole) is added asa solution in DMF (230 mL). The pH is adjusted to 7-8 by addition ofDIPEA (45 mL) and the mixture allowed to reach ambient temperature.After 2 hours, reaction is complete (HPLC analysis). The mixture isquenched into water (2.5 L) and extracted with EtOAc (1 L). The aqueousphase is back-extracted with EtOAc (0.3 L). The organic layers arecombined, washed with aqueous citric acid (5% w/w, 2×1 L), washed withaqueous NaHCO₃ (5% w/w, 2×1 L), and washed with water (2 L). The EtOAclayer is transferred to a 2L flask and heptane (500 mL) is added whilestirring to affect crystallization. The solids are collected by suctionon a Buchner funnel, washed with EtOAc/Heptane (2:1 v/v, 1 L) and driedto constant weight to yieldCBZ-PipBuen-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (143.2 g, 0.19 mole, 83%yield).

Elemental analysis: C₄₁ H₅₅ N₅ O₇ C: calc. 66.02; fd. 65.53, H, N. MassSpec: M_(calc) 745.91; M+1_(obsvd) 746 ¹ H NMR (δ vs TMS, CDCl₃): 0.86qd (1H); 0.98, qd (1H); 1.16, t (2H), 1.24, dt (6H); 1.37, m (1H);1.64-1.78, m (4H); 1.86, qd (1H); 2.2 bd (4H); 2.35, m (1H); 2.4, m(2H); 2.74, dd (1H); 3.07, m (4H); 3.52, d, (1H); 3.85, d (1H); 4.12, q(1H); 4.49, qd (1H); 4.68, dt (1H); 5.07, d (1H); 5.14, s (1H); 5.16, d(1H); 5.22, t (2H); 6.45, s (1H); 7.28, d (1H); 7.26, s (5H); 7.35, s(5H); 7.56, d (1H) ¹³ C NMR (δ vs TMS, CDCl₃): 14.15, 22.68, 24.95,25.61, 26.03, 26.45, 28.20, 31.71, 32.89, 33.80, 33.89, 34.00, 35.63,38.37, 44.79, 45.13, 45.65, 50.23, 51.34, 60.40, 66.87, 67.06, 76.50,77.13, 77.77, 122.46, 126.88, 127.80-128.60, 135.15, 155.19, 170.11,170.20, 171.61, 173.76, 175.35.

EXAMPLE 11 Preparation of Hygroscopic Crystalline Form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (Alternate Step 5 of Scheme II)

CBZ-PipBuen-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (140 g 0.19 mole),ammonium formate (61 g, 0.96 mole) and 10% Pd/C (50% wet, degussa type,28 g) are added to a 5 L jacketed flask. EtOH (200 proof, 1260 mL),iPrOH (70 mL) and water (DI, 70 g) are added. This mixture is heated to40-50° C., and stirred until HPLC indicates complete reaction (5 hours).The mixture is cooled to room temperature and filtered to remove thecatalyst. The resulting solution is heated to 40-50° C. and acetone(˜equal volume vs. filtered solution) added, allowing the solution tocool to 35-40° C. Seeds ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]--(L)-aspartyl]-(L)-β-cyclohexylalanine amide are added to the mixture and hygroscopic formofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide crystallizes therefrom while cooling to room temperature (severalhours). The solids are collected by suction on a Buchner funnel under ablanket of nitrogen, the cake is washed with acetone and air dried toconstant weight to yieldN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]--(L)-aspartyl]-(L)-.beta.-cyclohexyl-alanineamide (84.3 g, 0.16 mole, 84.8% yield, >95A %).

EXAMPLE 12 Concatenated Preparation ofTFA-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (Alternate of Steps 1-3 of SchemeII)

A 500 mL flask fitted with a temperature probe is charged withBOC-N(Et)-Gly (20.3 g, 0.1 mole), N-hydroxysuccinimide (115.0 g, 0.1mole) and dichloromethane (200 mL). The mixture is stirred at moderatespeed and to the resulting solution is added DCC (20.6 g, 0.1 mole) inone portion as a solid. This solution is stirred for 1 hour during whicha small exotherm is noticed (temperature rise from 20° C. to 28° C.) andDCU precipitates. The resulting suspension is vacuum filtered using aBuchner funnel equipped with a Whatman #1 filter paper. The cake iswashed with dichloromethane (2×25 mL). The filtrates are returned to theoriginal 500 mL flask and then (L)Asp(OBzl) (22.3 g, 0.1 mole), NMM(33.8 mL, 0.3 mole) and DMF (80 g, 1.01 mole) are added successively.After being stirred for 2 hours at room temperature, formation ofBOC-N(Et)Gly-(L)-Asp(OBzl) is complete (HPLC monitoring). The reactionmixture is poured into an extraction funnel containing ice water (100mL). The mixture is acidified with HCl (36%, 25 mL) until pH 1. Thelayers are split and the dichloromethane layer is washed with ice water(100 mL) and the phases split (aq. phase pH 3-4). The dichloromethanelayer is returned to the original 500 mL flask which is chargedsuccessively with NH₂ -(L)-Cha-NH₂ (17 g, 0.1 mole)),N-hydroxysuccinimide (11.5 g, 0.1 mole) in one portion each as solids.After stirring for 2 hours at room temperature, the formation ofBOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ is complete (HPLC monitoring) andthe DCU is vacuum filtered using a Buchner funnel equipped with aWhatman #1 filter paper. The cake is washed with dichloromethane (2×25mL). The filtrate is transferred to an extraction funnel and washed withdeionized water (200 mL) containing N-methyl morpholine (15 mL, pH 8-9).The phases are split and the dichloromethane layer is again washed withwith water (DI, 2×150 mL). The dichloromethane phase is washed with 150mL of 1 N HCl (pH 1). The phases are split and the dichloromethane layeris washed with deionized water (200 mL, pH 3). The dichloromethanesolution of BOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ is returned to aclean 500 mL flask and then charged with TFA (100 mL). After beingstirred for 2 hours at room temperature, the formation ofTFA.HN(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ is complete (HPLC monitoring).The reaction mixture is distilled under vacuum to remove thedichloromethane and most of the TFA, then MTBE (500 mL) and seeds areadded to effect product crystallization. The mixture is vacuum filteredusing a Buchner funnel equipped with a Whatman #1 filter paper. The cakeis washed with MTBE (2×25 mL) and air-dried to affordTFA.HN(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂ (46.8 g, 81.5% yield) as a whitesolid (>97A % pure, <0.2A % D-Asp diast.).

EXAMPLE 13 Preparation of Stable Non-hygroscopic Crystalline Form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide

Method A. Static Conversion

Hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (7.45 Kg) is milled in a hammer mill. The resulting solid, 7.35kg, is placed in a stainless steel dryer tray (90×28 cm) and the tray iscovered with perforated aluminum foil. The tray is then sealed into ahumidity oven (LUNAIRE Humidity Cabinet model no. CEO 941W-3); the ovenis kept sealed throughout the form conversion process except to removesamples for analysis. The oven is adjusted to 40% RH and 60° C. and keptat those levels for 1 hour. The humidity oven is then adjusted to 80%RH/60° C. and held at those levels for 12 hours. A sample is removedafter 18 hours at 80% RH/60° C. and checked by X-Ray powder diffractionanalysis to assess the conversion to the non-hygroscopic crystallineform ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide. The humidity oven is resealed and adjusted to 40% RH/60° C. andheld there for 2 hours. The oven is readjusted to ambient conditions,then the tray is then removed from the oven and the non-hygroscopiccrystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide is yielded (7.2 kg, 96.6% yield). Confirmation of the conversionis determined by an X-Ray powder diffraction graph (FIG. 1). The X-raypowder diffraction is also tabularized as a function of increasing orderof the angle of diffraction (2θ) corresponding to the interplanardistance of the crystal (d) in angstrom units (Å), counts per second(Cps) and relative peak intensity (%) (Table 1).

                  TABLE                                                           ______________________________________                                        N-       2θ                                                             d---                                                                          Cps---                                                                        %---                                                                          ______________________________________                                        1        5.065   17.4314     86.00  5.82                                      2        6.323   13.9672     248.00 16.78                                     3        7.518   11.7489     221.00 14.95                                     4        8.163   10.8222     496.00 33.56                                     5        8.780   10.0633     155.00 10.49                                     6        10.383  8.5125      218.00 14.75                                     7        11.351  7.7886      112.00 7.58                                      8        12.596  7.0218      999.00 67.59                                     9        13.858  6.3852      316.00 21.38                                     10       15.191  5.8274      1338.00                                                                              90.53                                     11       16.476  5.3759      481.00 32.54                                     12       16.745  5.2901      556.00 37.62                                     13       17.980  4.9294      679.00 45.95                                     14       18.572  4.7735      1079.00                                                                              73.00                                     15       18.799  4.7165      1230.00                                                                              83.22                                     16       19.147  4.6315      1229.00                                                                              83.15                                     17       l9.619  4.5211      1380.00                                                                              93.37                                     18       20.200  4.3924      1246.00                                                                              84.30                                     19       20.466  4.3360      1478.00                                                                              100.00                                    20       20.870  4.2528      1088.00                                                                              73.61                                     21       21.625  4.1061      584.00 39.51                                     22       22.088  4.0210      891.00 60.28                                     23       22.840  3.8903      613.00 41.47                                     24       23.947  3.7129      597.00 40.39                                     25       24.569  3.6203      680.00 46.01                                     26       25.608  3.4757      506.00 34.24                                     27       27.015  3.2978      1100.00                                                                              74.42                                     28       27.837  3.2022      420.00 28.42                                     29       27.967  3.1877      400.00 27.06                                     30       29.255  3.0502      536.00 36.27                                     31       29.689  3.0066      603.00 40.80                                     32       30.665  2.9130      518.00 35.05                                     33       31.318  2.8538      451.00 30.51                                     34       31.894  2.8036      533.00 36.06                                     35       33.370  2.6829      518.00 35.05                                     36       33.562  2.6679      552.00 37.35                                     37       33.919  2.6407      581.00 39.31                                     38       34.840  2.5730      561.00 37.96                                     39       35.789  2.5069      559.00 37.82                                     40       35.940  2.4967      560.00 37.89                                     41       36.780  2.4416      740.00 50.07                                     42       37.042  2.4249      736.00 49.80                                     43       37.959  2.3684      683.00 46.21                                     44       39.017  2.3066      643.00 43.50                                     ______________________________________                                    

Method B. Dynamic Conditions

a. Form Conversion

Hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (50 g) is placed in a 400 mL graduated cylinder (bed height 6 cm)on a ring stand and equipped with a mechanical stirrer. The apparatus isplace in a humidity-controlled oven (oven(LUNAIRE Humidity Cabinet modelno. CEO 941W-3). Agitation is set at 275 rpm, and the temperature and RHare adjusted over 30 minutes to 60° C. and 40%, respectively. Thecompound is held at these conditions for 1 hour, then conditions arechanged over 45 minutes to 80% RH/60° C. The compound is then held atthese conditions for 16 hours before the oven is reset to 40% RH/60° C.and held there for 3.25 hours. The compound is then allowed to return toambient conditions (bed height 4 cm), then removed from the cylinder toyield the non-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (yield >95%). Confirmation of the conversion is determined byX-Ray powder diffraction analysis (FIG. 2). The X-ray powder diffractionis also tabularized as a function of increasing order of the angle ofdiffraction (2θ) corresponding to the interplanar distance of thecrystal (d) in angstrom units (Å), counts per second (Cps) and relativepeak intensity (%) (Table 2).

                  TABLE 2                                                         ______________________________________                                        N-       2θ                                                             d---                                                                          Cps---                                                                        %---                                                                          ______________________________________                                        1        5.186   17.0268     196.00 8.43                                      2        6.371   13.8615     722.00 31.07                                     3        7.570   11.6689     516.00 22.20                                     4        8.232   10.7323     1094.00                                                                              47.07                                     5        8.817   10.0206     257.00 11.06                                     6        10.428  8.4761      365.00 15.71                                     7        11.377  7.7714      129.00 5.55                                      8        11.600  7.6223      117.00 5.55                                      9        12.667  6.9828      1805.00                                                                              77.67                                     10       13.913  6.3599      551.00 23.71                                     11       14.398  6.1468      178.00 7.66                                      12       15.226  5.844       2285.00                                                                              98.32                                     13       16.538  5.3557      861.00 37.05                                     14       16.773  5.2814      929.00 39.97                                     15       18.019  4.9190      1132.00                                                                              48.71                                     16       18.672  4.7483      1871.00                                                                              80.51                                     17       18.815  4.7125      2052.00                                                                              88.30                                     18       19.204  4.6178      2071.00                                                                              89.11                                     19       19.654  4.5132      2226.00                                                                              95.78                                     20       20.237  4.3845      1939.00                                                                              83.43                                     21       20.523  4.3240      2324.00                                                                              100.00                                    22       20.934  4.2400      1656.00                                                                              71.26                                     23       21.691  4.0938      923.00 39.72                                     24       22.143  4.0112      1411.00                                                                              60.71                                     25       22.910  3.8786      994.00 42.77                                     26       24.007  3.7037      964.00 41.48                                     27       24.642  3.6097      991.00 42.64                                     28       25.642  3.6097      991.00 42.64                                     29       27.070  3.2913      1687.00                                                                              72.59                                     30       27.855  3.2002      688.00 29.60                                     31       29.497  3.0258      843.00 36.27                                     32       29.497  3.0013      878.00 37.78                                     33       30.751  2.9051      809.00 34.81                                     34       31.916  2.8017      821.00 35.33                                     35       33.982  2.6360      882.00 37.95                                     36       35.200  2.5475      865.00 37.22                                     37       36.001  2.4926      841.00 36.19                                     38       36.927  2.4322      1106.00                                                                              47.59                                     39       38.389  2.3429      968.00 41.65                                     ______________________________________                                    

b. Form Conversion

Hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (370 g) is charged into a 2 L rotary evaporator flask. The flaskis placed on the rotary evaporator (Heidolph UV 2002) and lowered into apreheated (58° C.) bath (Heidolph MR 2002). The apparatus is placedunder a vacuum of 60 mBar using a vacuum pump (Divatrion DV1), thenvacuum is broken in a controlled fashion to admit a humid atmospherecreated in a separate, heated, water-containing flask. The admission ofhumid atmosphere is controlled by a humidity controlling apparatus(Vausalo Humiditique and Temperature Traumettor) so as to achieve a RHof 79% within the apparatus (130-180 mBar internal pressure). The rotaryevaporator vessel is then rotated at 145-160 revolutions per minute overa period of 5 hours while the heating bath is maintained at ˜60° C. andthe RH maintained within the vessel at 71-79%. Vacuum is then broken tonitrogen, the vessel and its contents allowed to cool to ambienttemperature, and the product is removed to yield the non-hygroscopiccrystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide. A second 317 g lot of the hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide is similarly treated to provide the non-hygroscopic crystallineform ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexyl-alanineamide. Confirmation of the conversion is determined by X-Ray powderdiffraction analysis (FIG. 3). The two lots together afforded 667 g ofthe non-hygroscopic crystalline form ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide (97% yield overall). Confirmation of the conversion is determinedby X-Ray powder diffraction analysis (FIG. 3). The X-ray powderdiffraction is also tabularized as a function of increasing order of theangle of diffraction (2θ) corresponding to the interplanar distance ofthe crystal (d) in angstrom units (Å), counts per second (Cps) andrelative peak intensity (%) (Table 3).

                  TABLE 3                                                         ______________________________________                                        N-       2θ                                                             d---                                                                          Cps---                                                                        %---                                                                          ______________________________________                                        1        5.124   17.2309     180.00 10.17                                     2        6.328   13.9565     408.00 23.05                                     3        7.574   11.6623     305.00 17.23                                     4        8.191   10.7851     556.00 31.41                                     5        8.797   10.0432     166.00 9.38                                      6        10.398  8.5004      244.00 13.79                                     7        12.628  7.0040      1198.00                                                                              67.68                                     8        13.871  6.3791      353.00 19.94                                     9        15.218  5.8172      1543.00                                                                              87.18                                     10       15.723  5.6317      187.00 10.56                                     11       16.538  5.3558      589.00 33.28                                     12       16.751  5.2882      621.00 35.08                                     13       18.024  4.9175      869.00 49.10                                     14       18.640  4.7563      1156.00                                                                              65.31                                     15       18.809  4.7141      1241.00                                                                              70.11                                     16       19.191  4.6210      1521.00                                                                              85.93                                     17       19.659  4.5120      1413.00                                                                              79.83                                     18       20.865  4.4064      1303.00                                                                              73.62                                     19       20.495  4.3299      1770.00                                                                              100.00                                    20       20.865  4.2539      1120.00                                                                              63.28                                     21       21.616  4.1077      683.00 38.59                                     22       22.113  4.0166      919.00 51.92                                     23       22.950  3.8719      697.00 39.38                                     24       24.117  3.6871      659.00 37.23                                     25       24.618  3.6132      716.00 40.45                                     26       25.644  3.4709      662.00 37.40                                     27       26.297  3.3862      486.00 27.46                                     28       27.052  3.2934      1270.00                                                                              71.75                                     29       27.960  3.1885      518.00 29.27                                     30       29.640  3.0115      705./00                                                                              39.38                                     31       30.744  2.9058      695.00 39.27                                     32       33.465  2.6755      697.00 39.38                                     33       33.840  2.6467      764.00 43.16                                     34       35.812  2.5053      736.00 41.58                                     35       36.811  2.4396      858.00 48.47                                     36       37.076  2.4228      919.00 51.92                                     37       38.185  2.3549      870    49.15                                     38       39.622  2.2728      882.00 49.83                                     ______________________________________                                    

EXAMPLE 14 X-Ray Powder Diffraction Graphs of a Sample ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide in its Hygroscopic Crystalline Form and its ConvertedNon-hygroscopic Crystalline Form

A sample of hygroscopic crystallineN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide is prepared as in Example 5 or 11, and converted to thecorresponding non-hygroscopic crystalline form according to a method ofExample 13. The x-ray powder diffraction graphs of the hygroscopiccrystalline form and non-hygroscopic crystalline form are shownrespectively in FIGS. 4 and 5. The X-ray powder diffraction graphs forthe hygroscopic crystalline form and non-hygroscopic crystalline formare also tabularized as a function of increasing order of the angle ofdiffraction (2θ) corresponding to the interplanar distance of thecrystal (d) in angstrom units (Å), counts per second (Cps) and relativepeak intensity (%) in Table 4 and Table 5, respectively.

                  TABLE 4                                                         ______________________________________                                        N-       2θ                                                             d---                                                                          Cps---                                                                        %---                                                                          ______________________________________                                        1        5.073   17.4037     1487.00                                                                              86.50                                     2        6.451   13.6905     447.00 26.00                                     3        7.837   11.2712     411.00 23.91                                     4        8.491   10.4049     602.00 35.02                                     5        9.699   9.1119      93.00  5.41                                      6        10.488  8.4278      421.00 24.49                                     7        11.570  7.6423      92.00  5.35                                      8        12.550  7.0474      411.00 23.91                                     9        13.576  6.5168      760.00 44.21                                     10       15.327  5.7763      606.00 35.25                                     11       15.790  5.6080      456.00 26.53                                     12       16.179  5.4739      346.00 20.13                                     13       16.770  5.2824      938.00 54.57                                     14       17.085  5.1856      685.00 39.85                                     15       17.750  4.9927      924.00 53.75                                     16       18.151  4.8835      741.00 43.11                                     17       18.504  4.7909      593.00 34.50                                     18       19.323  4.5897      930.00 54.10                                     19       19.714  4.4996      792.00 46.07                                     20       20.545  4.3194      1719.00                                                                              100.00                                    21       21.388  4.1510      897.00 52.18                                     22       22.381  3.9691      373.00 21.70                                     23       22.870  3.8852      258.00 15.01                                     24       23.640  3.7604      563.00 32.75                                     25       23.841  3.7292      680.00 39.56                                     26       24.048  3.6976      623.00 36.24                                     27       24.746  3.5949      338.00 19.66                                     28       25.200  3.5311      366.00 21.29                                     29       25.792  3.4513      590.00 34.32                                     30       26.266  3.3901      731.00 42.52                                     31       26.959  3.3045      555.00 32.29                                     32       27.426  3.2494      769.00 44.74                                     33       27.967  3.1876      528.00 30.72                                     34       29.020  3.0744      771.00 44.85                                     35       29.922  2.9837      491.00 28.56                                     36       30.970  2.8851      384.00 22.34                                     37       31.552  2.8332      510.00 29.67                                     38       33.338  2.6854      627.00 36.47                                     39       34.838  2.5731      520.00 30.25                                     40       35.873  2.5012      653.00 37.99                                     41       36.107  2.4855      639.00 37.17                                     42       37.162  2.4174      683.00 39.73                                     43       38.509  2.3359      775.00 45.08                                     44       39.701  2.2684      784.00 45.61                                     ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        N-       2θ                                                             d---                                                                          Cps---                                                                        %---                                                                          ______________________________________                                        1        5.152   17.1371     123.00 7.34                                      2        6.386   13.8287     483.00 28.84                                     3        7.580   11.6540     389.00 23.22                                     4        8.225   10.7410     752.00 44.90                                     5        8.801   10.0390     180.00 10.75                                     6        10.408  8.4928      276.00 16.48                                     7        12.660  6.9863      1399.00                                                                              83.52                                     8        13.914  6.3594      391.00 23.34                                     9        15.251  5.8047      1675.00                                                                              100.00                                    10       16.541  5.3548      608.00 36.30                                     11       16.771  5.2818      652.00 38.93                                     12       18.047  4.9112      775.00 46.27                                     13       18.676  4.7472      1078.00                                                                              64.36                                     14       18.902  4.6910      1099.00                                                                              65.61                                     15       19.182  4.6231      1151.00                                                                              68.72                                     16       19.697  4.5035      1164.00                                                                              69.49                                     17       20.240  4.3838      1049.00                                                                              62.63                                     18       20.568  4.3147      1403.00                                                                              83.76                                     19       29.933  4.2403      1024.00                                                                              61.13                                     20       21.684  4.0951      569.00 33.97                                     21       22.122  4.0150      746.00 44.54                                     22       22.970  3.8685      564.00 33.67                                     23       24.080  3.6927      546.00 32.60                                     24       24.218  3.6720      556.00 33.19                                     25       24.694  3.6023      618.00 36.90                                     26       25.680  3.4662      510.00 30.45                                     27       26.400  3.3732      403.00 24.06                                     28       27.105  3.2871      1093.00                                                                              65.25                                     29       27.929  3.1920      450.00 26.87                                     30       29.360  3.0395      555.00 33.13                                     31       29.724  3.0031      595.00 35.52                                     32       30.340  2.9435      429.00 25.61                                     33       30.693  2.9105      552.00 32.96                                     34       31.353  2.8507      476.00 28.42                                     35       31.822  2.8098      531.00 31.70                                     36       32.006  2.7940      545.00 32.54                                     37       32.885  2.7213      485.00 28.96                                     38       33.508  2.6722      547.00 32.66                                     39       34.040  2.6316      606.00 36.18                                     40       34.839  2.5730      580.00 34.63                                     41       35.998  2.4928      596.00 35.58                                     42       36.680  2.4480      629.00 37.55                                     43       36.948  2.4309      727.00 43.40                                     44       37.197  2.4152      703.00 41.97                                     45       39.602  2.2739      697.00 41.61                                     ______________________________________                                    

EXAMPLE 15 Isothermal Microcalorimetric Experiments on Hygroscopic andNon-Hygroscopic Crystalline Forms ofN-[N-[N-(4-Piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide

Isothermal microcalorimetry experiments on hygroscopic andnon-hygroscopic crystalline forms ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide are performed on a Thermometric® Thermal Activity Monitor (TAM).Solid state conversions of the different crystalline forms are studiedby exposing the forms to different humidities or solvent vapors atdifferent temperatures. The saturated salt solutions used to obtaindifferent humidities were: KCl (80% RH), NaCl (75% RH), and NaBr (65%RH). Approximately 100 mg quantities of the forms are weighted into aTAM glass ampule and a microhygrostate containing a saturated saltsolution (with excess solid) or an organic solvent is placed inside theampule. The ampule is sealed, equilibrated to the temperature of theexperiment, and lowered into the measuring position in the TAM. Anidentical system containing washed sea sand, in place of the formsubject to testing, is placed on the reference side. Output power (μW)is measured as a function of time (FIGS. 6-8).

EXAMPLE 16 Moisture Sorption Isotherms of Hygroscopic andNon-Hygroscopic Crystalline Forms ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide

Moisture Sorption Isotherms of hygroscopic and non-hygroscopiccrystalline forms ofN-[N-[N-(4-piperdin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-β-cyclohexylalanineamide are obtained on a VTI MB300G moisture balance. The experiments areconducted either by subjecting approximately 15 mg of the subjectcrystalline form to increasing and decreasing steps of % RH andfollowing the weight gain (at each equilibrium step) as a function of %RH (FIG. 9) or by holding the subject crystalline form at constanthumidity and following the weight gain as a function of time.

The compound of formula II exhibits useful pharmacological activity andaccordingly are incorporated into pharmaceutical compositions and usedin the treatment of patients suffering from certain pathologicalconditions.

The present invention is also directed to a method for the treatment ofa patient suffering from, or subject to, conditions which can beameliorated or prevented by the administration of an inhibitor ofplatelet aggregation by inhibiting fibrinogen binding to activatedplatelets and other adhesive glycoproteins involved in plateletaggregation and blood clotting. Furthermore, the present invention isdirect to a method for preventing or treating thrombosis associated withcertain disease states, such as myocardial infarction, stroke,peripheral arterial disease and disseminated intravascular coagulationin humans and other mammals.

Reference herein to treatment should be understood to includeprophylactic therapy as well as treatment of established conditions.

The present invention also includes within its scope a pharmaceuticalcomposition which comprises a pharmaceutically acceptable amount of atleast one compound of formula I in association with a pharmaceuticallyacceptable carrier or excipient.

In practice compounds or compositions for treating according to thepresent invention may administered by any suitable means, for example,by topically, inhalation, parenterally, rectally or orally, but they arepreferably administered orally.

The compound of formula II may be presented in forms permittingadministration by the most suitable route and the invention also relatesto pharmaceutical compositions containing at least one compoundaccording to the invention which are suitable for use in human orveterinary medicine. These compositions may be prepared according toconventional methods, using one or more pharmaceutically acceptableadjuvants or excipients. The adjuvants comprise, inter alia, diluents,sterile aqueous media and the various non-toxic organic solvents. Thecompositions may be presented in the form of tablets, pills, capsules,granules, powders, aqueous solutions or suspensions, injectablesolutions, elixirs, syrups and the like, and may contain one or moreagents chosen from the group comprising sweeteners, flavorings,colorings, stabilizers or preservatives in order to obtainpharmaceutically acceptable preparations.

The choice of vehicle and the content of active substance in the vehicleare generally determined in accordance with the solubility and chemicalproperties of the product, the particular mode of administration and theprovisions to be observed in pharmaceutical practice. For example,excipients such as lactose, sodium citrate, calcium carbonate, dicalciumphosphate and disintegrating agents such as starch, alginic acids andcertain complex silica gels combined with lubricants such as magnesiumstearate, sodium lauryl sulfate and talc may be used for preparingtablets. To prepare a capsule, it is advantageous to use lactose andhigh molecular weight polyethylene glycols. When aqueous suspensions areused they may contain emulsifying agents or agents which facilitatesuspension. Diluents such as sucrose, ethanol, polyethylene glycol,propylene glycol, glycerol and chloroform or combinations thereof can beemployed as well as other materials.

For parenteral administration, emulsions, suspensions or solutions ofthe compounds according to the invention in vegetable oil, for examplesesame oil, groundnut oil or olive oil, or aqueous-organic solutionssuch as water and propylene glycol, injectable organic esters such asethyl oleate, as well as sterile aqueous solutions of thepharmaceutically acceptable salts, are used. The solutions of the saltsof the products according to the invention are also useful foradministration by intramuscular or subcutaneous injection. The aqueoussolutions, also comprising solutions of the salts in pure distilledwater, may be used for intravenous administration with the proviso thattheir pH is suitably adjusted, that they are judiciously buffered andrendered isotonic with a sufficient quantity of glucose or sodiumchloride and that they are sterilized by heating, irradiation and/ormicrofiltration.

Topical administration, gels (water or alcohol based), creams orointments containing compounds of the invention may be used. Compoundsof the invention may be also incorporated in a gel or matrix base forapplication in a patch, which would allow a controlled release ofcompound through transdermal barrier.

Solid compositions for rectal administration include suppositoriesformulated in accordance with known methods and containing at least onecompound of formula II.

The percentage of active ingredient in a composition according to theinvention may be varied such that it should constitutes a proportion ofa suitable dosage. Obviously, several unit dosage forms may beadministered at about the same time. A dose employed may be determinedby a physician or qualified medical professional, and depends upon thedesired therapeutic effect, the route of administration and the durationof the treatment, and the condition of the patient. The dosage regimenin carrying out the method of this invention is that which insuresmaximum therapeutic response until improvement is obtained andthereafter the minimum effective level which gives relief. In general,the oral dose may be between about 0.1 mg/kg and about 100 mg/kg,preferably between about 0.1 mg/kg to 20 mg/kg, and most preferablybetween about 1 mg/kg and 20 mg/kg, and the i.v. dose about 0.1 μg/kg toabout 100 μg/kg, preferably between about 0.1 mg/kg to 50 mg/kg. In eachparticular case, the doses are determined in accordance with the factorsdistinctive to the patient to be treated, such as age, weight, generalstate of health and other characteristics which can influence theefficacy of the compound according to the invention.

Furthermore, a compound of formula II may be administered as frequentlyas necessary in order to obtain the desired therapeutic effect. Somepatients may respond rapidly to a higher or lower dose and may find muchweaker maintenance doses adequate. For other patients, it may benecessary to have long-term treatments at the rate of 1 to 4 oral dosesper day, preferably once to twice daily, in accordance with thephysiological requirements of each particular patient. Generally, theactive product may be administered orally 1 to 4 times per day. Ofcourse, for other patients, it will be necessary to prescribe not morethan one or two doses per day.

A compound of formula II exhibits marked phamacological activitiesaccording to tests described in the literature which tests results arebelieved to correlate to phamacological activity in humans and othermammals. The following pharmacological in vitro and in vivo test resultsare typical for characterizing a compound of formula II.

The following pharmacologic tests evaluate the inhibitory activity of acompound of formula II on fibrinogen-mediated platelet aggregation,fibrinogen binding to thrombin-stimulated platelets, and inhibition ofADP-induced ex-vivo platelet aggregation, and results of these testscorrelate to the in-vivo inhibitory properties of a compound of formulaII.

The Platelet Aggregation Assay is based on that described in Blood 66(4), 946-952 (1985). The Fibrinogen-Binding Assay is essentially that ofRuggeri, Z., et al., Proc. Natl. Acad. Sci. USA 83, 5708-5712 (1986) andPlow, E. F., et al., Proc. Natl. Acad. Sci., USA 82, 8057-8061 (1985).The Inhibition of ADP-Induced ex-vivo Platelet Aggregation assay isbased on that of Zucker, "Platelet Aggregation Measured by thePhotoelectric Method", Methods in Enzymology 169, 117-133 (1989).

Platelet Aggregation Assay

Preparation of Fixed-Activated Platelets

Platelets are isolated from human platelet concentrates using thegel-filtration technique as described by Marguerie, G. A. et al., J.Biol. Chem. 254, 5357-5363 (1979) and Ruggeri, Z. M. et al., J. Clin.Invest. 72, 1-12 (1983). The platelets are suspended at a concentrationof 2×10⁸ cells/mL in a modified calcium-free Tyrode's buffer containing127 mM sodium chloride, 2 mM magnesium chloride, 0.42 mM Na₂ HPO₄, 11.9mM NaHCO₃, 2.9 mM KCl, 5.5 mM glucose, 10 mM HEPES, at a pH of 7.35 and0.35% human serum albumin (HSA). These washed platelets are activated byaddition of human a-thrombin at a final concentration of 2 units/mL,followed by thrombin inhibitor I-2581 at a final concentration of 40 μM.To the activated platelets is added paraformaldehyde to a finalconcentration of 0.50% and this incubated at room temperature for 30minutes. The fixed activated platelets are then collected bycentrifugation at 650×g for 15 minutes. The platelet pellets are washedfour times with the above Tyrode's-0.35% HSA buffer and resuspended to2×10⁸ cells/mL in the same buffer.

Platelet Aggregation Assay

The fixed activated platelets are incubated with a selected dose of thecompound to be tested for platelet aggregation inhibition for one minuteand aggregation initiated by addition of human fibrinogen to a finalconcentration of 250 μg/mL. A platelet aggregation profiler Model PAP-4is used to record the platelet aggregation. The extent of inhibition ofaggregation is expressed as the percentage of the rate of aggregationobserved in the absence of inhibitor. IC₅₀, i.e., the amount ofinhibitor required to reduce the aggregation rate by 50%, is thencalculated for each compound (see, for example, Plow, E. F. et al.,Proc. Natl. Acad. Sci., USA 82, 8057-8061 (1985)).

Fibrinogen-Binding Assay

Platelets are washed free of plasma constituents by the albumindensity-gradient technique of Walsh, P. N. et al., Br. J. Haematol.281-296 (1977), as modified by Trapani-Lombardo, V. et al., J. ClinInvest. 76, 1950-1958 (1985). In each experimental mixture platelets inmodified Tyrode's buffer (Ruggeri, Z. M. et al., J. Clin. Invest. 72,1-12 (1983)) are stimulated with human a-thrombin at 22-25° C. for 10minutes (3.125×10¹¹ platelets per liter and thrombin at 0.1 NIHunits/mL). Hirudin is then added at a 25-fold excess (unit/unit) for 5minutes before addition of the ¹²⁵ I-labeled fibrinogen and the compoundto be tested. After these additions, the final platelet count in themixture is 1×10¹¹ /L. After incubation for an additional 30 minutes at22-25° C., bound and free ligand are separated by centrifuging 50 μL ofthe mixture through 300 μL of 20% sucrose at 12,000×g for 4 minutes. Theplatelet pellet is then separated from the rest of the mixture todetermine platelet-bound radioactivity. Nonspecific binding is measuredin mixtures containing an excess of unlabeled ligand. When bindingcurves are analyzed by Scatchard analysis, nonspecific binding isderived as a fitted parameter from the binding isotherm by means of acomputerized program (Munson, P. J., Methods Enzymol. 92, 542-576(1983)). To determine the concentration of each compound necessary toinhibit 50% of fibrinogen binding to thrombin-stimulated platelets(IC₅₀), each compound is tested at 6 or more concentrations with ¹²⁵I-labeled fibrinogen held at 0.176 μmol/L (60 μg/mL). The IC₅₀ isderived by plotting residual fibrinogen binding against the logarithm ofthe sample compound's concentration.

Inhibition of ADP-Induced Ex-Vivo Platelet Aggregation

Experimental Protocol

Control blood samples are obtained 5-10 minutes prior to administrationof a test compound in mongrel dogs weighing from 10 to 20 Kg. Thecompound is administered intragasticly, via aqueous gavage, or orally,via gelatin capsule. Blood samples (5 ml) are then obtained at 30 minuteintervals for 3 hours, and at 6, 12, and 24 hours after dosing. Eachblood sample is obtained by venipuncture of the cephalic vein and iscollected directly into a plastic syringe containing one part 3.8%trisodium citrate to nine parts blood.

Ex Vivo Canine Platelet Aggregation

The blood samples are centrifuged at 1000 rpm for 10 minutes to obtainplatelet rich plasma (PRP). After removal of the PRP, the sample iscentrifuged for an additional 10 minutes at 2000 rpm to obtain plateletpoor plasma (PPP). Platelet count in the PRP is determined by using aCoulter Counter (Coulter Electronics, Hialeah, Fla.) If theconcentration of platelets in the PRP is greater than 300,000platelets/μL, then the PRP is diluted with PPP to adjust the plateletcount to 300,000 platelets/μL. Aliquots of PRP (250 μL) are then placedin siliconized glass cuvettes (7.25×55 mm, Bio/Data Corp., Horsham,Pa.). Epinephrine (final concentration of 1 μM) is then added to thePRP, which is incubated for one minute at 37° C. A stimulator ofplatelet aggregation, ADP at a final concentration of 10 μM, is thenadded to the PRP. Platelet aggregation is monitoredspectrophotometrically utilizing a light transmission aggregometer(Bio/Data Platelet Aggregation Profiler, Model PAP-4, Bio/Data Corp.,Horsham, Pa.). For the testing of a compound, the rate of change (slope)of light transmittance and the maximum light transmittance (maximumaggregation) is recorded in duplicate. Platelet aggregation data arereported as the percent decrease (mean±SEM) in slope or maximumaggregation as compared to data obtained from control PRP, which isprepared from blood samples obtained prior to administration of the testcompound.

A compound of formula II exhibits marked activity in the foregoing testsand is considered useful in the prevention and treatment of thrombosisassociated with certain disease states. Antithrombotic activity in theex vivo canine platelet aggregation assay is predictive of such activityin humans (see, for example, Catalfamo, J. L., and Dodds, W. Jean,"Isolation of Platelets from Laboratory Animals", Methods Enzymol. 169,Part A, 27 (1989)). Results of testing of a compound of formula II bythe above methods are presented in the Table 6 below. Also presented inthe table are comparative test results for 4--4 (Piperidyl)butanoylglycyl aspartyl tryptophan, i.e., the compound disclosed in EuropeanPatent Application Publication No. 0479,481.

                  TABLE 6                                                         ______________________________________                                                                  Inhibition of ADP-Induced                                   Inhibition        ex-vivo Platelet Aggregation                                of Fixed          % Inhibition of ex-vivo                             Compound                                                                              Platelet          Platelet Aggregation                                of Example                                                                            Aggregation                                                                             Dose    After Oral Administration                           Number  (IC.sub.50 μM)                                                                       (mg/kg) 1h   3h   6h   12h  24h                             ______________________________________                                        15      0.097     5       100  100  100  98   50                              (Compound                                                                             0.047     5       53   <20                                            of EPA                                                                        `481)                                                                         ______________________________________                                    

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects of the invention, andobtain the ends and advantages mentioned, as well as those inherenttherein. The compounds, compositions, and methods described herein arepresented as representative of the preferred embodiments, or intended tobe exemplary and not intended as limitations on the scope of the presentinvention.

What is claimed is:
 1. A compound which is non-hygroscopic crystallineN-[N-[N-(4-piperidin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-.beta.-cyclohexylalanineamide or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the compound according to claim 1 and apharmaceutically acceptable carrier.
 3. A method for preparingnon-hygroscopic crystallineN-[N-[N-(4-piperidin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-.beta.-cyclohexylalanineamide comprising exposing hygroscopic crystallineN-[N-[N-(4-piperidin-4-yl)butanoyl)-N-ethylglycyl]-(L)-aspartyl]-(L)-.beta.-cyclohexylalanineamide to relative humidities from about 40% to 100% and at about 20° C.to about 80° C.
 4. The method according to claim 3 wherein the exposingis a relative humidities from about 65% to about 80%.
 5. The methodaccording to claim 3 wherein the exposing is at about 40° C. to about80° C.
 6. The method according to claim 3 wherein the exposing iseffected under static conditions.
 7. The method according to claim 3wherein the exposing is effected under dynamic conditions.